Spy IA As A Diagnostic and Prognostic Marker Of Cancer

Field of the Invention

The present invention relates to the use of Spy IA as a marker, and in particular, to the use of Spy IA as a diagnostic and prognostic marker for cancer.

Background of the Invention

Members of the Speedy/RJNGO family are unique cyclin-like regulators of the cell division cycle. lnXenopus oocytes, X-Spyl was shown to prematurely activate CDK2 and CDKl, and to allow progression through the G ₂ /M checkpoint via activation of the MAPK pathway, thus promoting rapid oocyte maturation (6, 18). The human Spyl homologue SpylAl, herein referred to as Spy IA, is expressed constitutively in most human tissues; it shortens the Gl/S transition through activation of CDK2 and is essential for cell proliferation to occur (24). Activation of the CDKs by Spyl/RTNGO proteins is thought to occur in an atypical fashion, independent of cyclin binding and in the absence of CDK phosphorylation within the T-loop (16). In addition, Spy IA can prevent the inhibitory effects of the CDK inhibitor, p27 ^KlP1 (p27) by directly promoting p27 degradation (19, 25). SpylA-induced proliferation requires endogenous p27, but whether the direct interaction between Spy IA and p27 is required for all Spyl A- mediated proliferative effects is not known. Research also shows that Spyl A plays a role in the DNA damage response, functioning to enhance cell survival and promote cell proliferation in lieu of apoptosis (2, 8). Importantly, recent observations have demonstrated that Spyl A is capable of promoting precocious development and tumorigenesis in the mammary gland and that Spyl A protein levels are implicated in invasive ductal carcinoma of the breast (36). Hence, determining how Spy IA protein levels are regulated may reveal novel information regarding the dynamics of cell cycle control during normal and abnormal growth conditions. Cyclin proteins are tightly regulated temporally and spatially, controlled on a fundamental level by the ubiquitin-proteasome system (UPS). The UPS is the primary mechanism involved in the selective degradation of intracellular and membrane-bound proteins, and aberrations in this critically important system are correlated to many diseases such as breast cancer (4, 27). The ubiquitination process involves the conjugation of ubiquitin, a highly conserved protein of 76 amino acids, to a substrate protein; this event can signal the substrate for degradation by the proteasome (14). Ubiquitination is orchestrated by members of three classes of enzymes: the ubiquitin-activating enzyme El, the ubiquitin- conjugating enzyme E2, and the ubiquitin-protein ligase E3. The El first forms a thiolester bond between a cysteine residue on itself and a glycine residue on the C-terminal of ubiquitin, which is followed by the transfer of ubiquitin to a cysteine residue on an E2 enzyme. Another transfer of the ubiquitin polypeptide then takes place, resulting in a thiolester bond between ubiquitin and a cysteine residue of an E3 enzyme. The E3 enzyme can then catalyze the formation of a bond between the C-terminal glycine of ubiquitin and either a lysine residue on the target protein resulting in monoubiquitination, or a lysine residue to a previously added ubiquitin molecule creating a chain of ubiquitin molecules known as polyubiquitination (13, 27). Single-site monoubiquitination, multiple sites of monoubiquitination and K63-linked ubiquitin chains have non-proteolytic regulatory roles involved in activities such as DNA repair, transcription, endocytosis, and protein trafficking. On the other hand, polyubiquitination at K48 of ubiquitin targets the substrate protein for degradation by the 26S proteasome (30). Prior to ubiquitination, the substrate protein is usually modified to accommodate recognition by the E3, often by phosphorylation (4).

X-Spyl is regulated in Xenopus oocytes by the UPS system and the activity of two different ubiquitin ligases which function at different stages of the cell cycle: SCF ^βTrCP and Siah-2 (10). Prior to meiosis, G2 arrest of the oocyte is maintained by SCF ^|3TrCP" mediated cleavage of the C-terminal of X-Spyl ; this is initiated by phosphorylation on S233 and S237 by protein kinase A (PKA) and glycogen synthase (kinase-3β). It is believed that the processed fragment of X- Spy 1 may inhibit meiotic maturation either by forming inactive heterodimers with unprocessed copies of X-Spyl, or by competitively interacting with CDKs. On the other hand, Siah-2 has been shown to be responsible for leading to the subsequent degradation of phosphorylated X-Spyl between meiosis I and meiosis II. It was further demonstrated that timely degradation of X-Spyl during the transition to meiosis II was essential for preventing DNA synthesis during meiosis. In mammals, SpylA mRNA is known to be up-regulated during Gj/S; however whether this protein is also regulated via protein degradation is currently not known (24). Solomon et. al. have suggested that the N-terminal region of SpylA may be involved in protein stability, however data in this regard has not been presented nor has this been further explored (5). Based on the cyclin-like function of SpylA, and the known regulation of X-Spyl, it is a valid hypothesis that SpylA may also be subject to ubiquitin-mediated proteolysis.

Summary of the Invention

In one aspect, the present invention provides a method diagnosing cancer, said method comprising: determining the relative amount of SpylA protein in a sample cell; determining the relative amount of SpylA protein in a normal cell; and comparing the relative amount of SpylA protein in the sample cell to the relative amount of protein in the normal cell.

In another aspect, the present invention provides a method of diagnosing cancer, said method comprising: determining the relative amount of Nedd4 protein in a sample cell; determining the relative amount of Nedd4 protein in a normal cell; and comparing the relative amount of Nedd4 protein in the sample cell to the relative amount of Nedd4 protein in the normal cell.

In another aspect, the present invention provides a method of treating or preventing cancer, said method comprising phosphorylating one or more amino acid residues in a SpylA protein in a target cell. In this publication, we demonstrate that Spy IA degradation occurs via ubiquitin-mediated proteolysis following entry into mitosis but prior to the anaphase transition. We determine that the N-terminal 1-57 amino acids within SpylA are essential to support regulated degradation of the protein. We further resolve that the E3 ligase, Nedd4, is capable of binding to SpylA during G2/M phase of the cell cycle, and that dominant negative forms of Nedd4 reduce ubiquitination and degradation of SpylA. Additionally, we identify 3 key amino acids within the N-terminal region of SpylA: Tl 5, S22 and T33, which are essential for SpylA degradation. Collectively, our data demonstrates that SpylA, like the cyclin family with which it resembles, is tightly regulated at the protein level. Importantly, we provide a novel link between SpylA and Nedd4, two proteins previously implicated in tumorigenesis. Additionally we show that non- degradable forms of SpylA do not prevent cell cycle progression and instead promote cell proliferation, further implicating that SpylA may contribute to uncontrolled growth in tumorigenesis.

Brief Description of the Drawings

Figure. 1. SpylA protein is degraded in a cell cycle dependent fashion.

(A) MCF7 cells were either untreated (Cntl), blocked by serum starvation (SS) or blocked and then released into media containing serum and nocodozole (NT) and analyzed by flow cytometry. Top panel shows flow cytometry profiles and the bottom panel shows % of cells in each phase of the cell cycle as determined by CPX analysis. (B) Cell lysates from each population described in A were lysed and analyzed using 10% SDS PAGE followed by immunoblotting with SpylA antibody (top panel), Cyclin E as cell cycle marker (middle panel) and Actin (lower panel). (C) 293T cells were either untreated (Cntl), blocked by double thymidine block (TB) or blocked and then released into media containing serum and nocodazole (NT) and analyzed by flow cytometry. Top panel shows flow cytometry profiles and the bottom panel shows % of cells in each phase of the cell cycle as determined by CPX analysis. (D) Cell lysates from each population described in C were lysed and analyzed using 10% SDS PAGE followed by immunoblotting with SpylA antibody (top panel) and Actin as a loading control (lower panel). (E) 293T cells were transfected with Cyclin Bl wild-type (CycBl), empty vector negative control (GFP) and a non-degradable cyclin Bl (CycB-D). Cell lysates from untransfected (Cntl) or transfected cells were analyzed with 10% SDS PAGE followed by immunoblotting with SpylA antibody.

Figure 2. SpylA degradation is proteosome dependent. (A) 293T cells were used for an in vivo ubiquitination assay. Cells were transfected with HA ubiquitin (Ha-Ub), Myc-SpylA-PCS3 (Myc-Spy 1 A) and PCS3 empty vector (Myc-Cntl). An equal amount of protein was immunoprecipitated for Myc and analyzed using SDS-PAGE followed by immunoblotting with HA antibody. Ha- Ub-Spyl A is depicted in the upper panel. The membrane was stripped and then re-probed with monoclonal Myc antibody (lower panel). (B) 293T cells were treated with a calpain inhibitor (LLNL; lane 3) and a proteasome inhibitor (MGl 32; lane 4) as well as the vehicle control for LLNL (ETOH; lane 2) and the vehicle control for MG132 (DMSO; lane 1). Equal amount of protein was analyzed with 10% SDS-PAGE followed by immunoblotting with SpylA antibody (upper panel) and Actin as a loading control (lower panel).

Figure 3. SpylA degradation relies on the N-terminal region. (A) A schematic diagram for the different SpylA truncation mutants is depicted and restriction sites used for cleavage of the region are indicated. (B) 293T cells were transfected with Myc-SpylA-PCS3 (wt; lane 1) or the different truncation mutant TMA-TMZ (A-Z; lanes 3-7) or PCS3 empty vector (-ve; lane 2). Transfected cells were treated with 70 ng of nocodazole for 16 hrs. The cells were lysed and subjected to 10% SDS-PAGE followed by immunoblotting with Myc antibody (upper panel), or Actin as a loading control (lower panel). (C) 293 T cells were transfected with Myc-SpylA-PCS3 (wt and -ve; lanes 1 & 2) and TMA-TMZ (A- Z; lanes 3-7). Cells were either treated with orthophosphoric acid ³² P (lanes 1, 3- 7) or left untreated (-ve; lane 2). The cells were lysed and an equal amount of protein was immunoprecipitated with Myc antibody. The immunoprecipitates were analyzed with 10% SDS-PAGE and analyzed by phosphoimager analysis. Upper panel shows phosphorylated c-Myc band, and middle panel shows c-Myc immunoblot. Lower panel shows quantified analysis of the blot. (D) Alignment of SpylA and X-Spy 1 N-terminal region (top panel) and C-terminal region (lower panel) are depicted. Regions known to play a role in the degradation of each are indicated in bold. Phosphorylation sites known to direct X-Spy 1 processing at T8 and S12 are also depicted by +. Essential residues for mediating the X-Spy 1- _SCF ^β τ ^rc _{P binding5 S} 233 _ari d S237, are also depicted by _\ The essential phosphorylation site for mediating Siah-2 binding, S243, is also depicted by *. The potential PPxxxxY binding region for WW domain proteins within SpylA is also shaded in grey. Potential phosphorylation sites within the N-terminal region of SpylA are also indicated by amino acid numbers above the sequence.

Figure 4. Nedd4 is the ubiquitin ligase for SpylA. (A) 293T cells were transfected with empty vectors (PCS3 or PCEP), Nedd4-PCEP (Nedd4) or Myc- SpylA-PCS3 (Myc- SpylA). Equal amounts of protein were immunoprecipitated with Myc antibody and then the precipitates were analyzed using 10% SDS

PAGE followed by immunoblotting with Nedd4 antibody (upper panel). Lysates from NlH 3T3 cells served as positive control for Nedd4 expression (+; lane 5). The membrane was stripped and re -probed with Myc antibody (lower panel). (B) 293T cells were transfected with empty vectors (PCS3 or PCEP), Nedd4-PCEP (Nedd4), Myc-Spyl A-PCS3 (Myc-SpylA) or Nedd4-PCEP Dominant Negative (Nedd4 ^DN ). Equal amounts of protein were immunoprecipitated with Myc antibody and analyzed using 10% SDS PAGE. Immunoblotting with HA antibody detected HA-Ub-tagged SpylA (Ha-Ub-Spy IA; upper panel). The membrane was stripped and re -probed with Myc antibody (lower panel). Densitometry was carried out using AlphaEaseFC software. (C) 293T cells were transfected with PCEP empty vector control (lane 1) and Nedd4-PCEP (Nedd4; lanes 2 & 3). Cells were either treated with MGl 32 (lane 2) or DMSO (lanes 1 & 3). Cell lysates were analyzed by 10% SDS PAGE followed by immunoblotting with SpylA antibody (upper panel). The membrane was stripped and re-probed with Actin antibody (lower panel). Densitometry was carried out using AlphaEaseFC software.

Figure 5. Phosphorylation on T15, T33 and S22 is needed for SpylA degradation. 293T cells were transfected with SpylA wild-type (WT), SpylA- Tl 5A (Tl 5A), SpylA-T33A (T33A), SpylA-S22A (S22A). (A) Cells were treated with 70 ng Nocadazole for 16 hrs. (G2 population; left panels) or untreated control (Asynchronous population; right panel). Half of the population was kept for flow cytometry analysis (see D) and the remainder were lysed and subjected to 10% SDS-PAGE followed by immunoblotting with Myc antibody (SpylA; upper panels) and Actin as a loading control (lower panel). (B) Transfected cells were co-transfected with Ha-Ub followed by treatment with MG 132 for 16 hrs. Lysates were immunoprecipitated with Myc and immunoblotted for Ha (Ha- Ub- Spyl A; upper panel), Nedd4 (middle panel) or Myc (lower panel). (C) Triplicate transfections were counted for alive and dead cells at 36 hrs. post transfection using trypan blue exclusion. Counts from the 3 separate transfections were used for statistical analysis. Error bars reflect standard deviation and a standard T-test was performed assuming equal variance. Statistical data shown reflects comparisons between the WT transfected cells and mutant transfected cells which were * p. 0.05, ** 135_ 0.01. (D) Cells from (A) were analyzed by flow cytometry. CPX analysis was carried out to determine the % of cells in each population. These numbers are depicted above the schematic of the cell cycle profiles.

Figure 6 illustrates example data of Spyl levels detected in tumour tissue vs control from a tissue microarray detection and quantification of Spyl protein levels.

Figure 7 illustrates Spyl levels in tumour tissue vs control from a tissue protein extraction and Western Blot protocol for SpylA protein levels.

Figure 8 illustrates: (A) expression of Spyl in proliferative mammary tissues (pregnancy) as well as regenerating mammary tissues (involution); (B) Western Blot analysis of the same tissues; (C) RT-PCR analysis of tissues. Figure 9 illustrates: (A) A schematic diagram for the different Spy IA deletion mutants is depicted and restriction sites used for cleavage of the region are indicated. (B) 293 cells were transfected with Myc-Spyl A-PCS3 (wt) or the different deletion mutant DMA-DMZ (A-Z). Transfected cells were treated with nocodazole (left hand panels; synchronous) or no treatment (right hand panels; asynchronous) for 16 hrs. post transfection. Lysates were immunoblotted with α- Myc or oc-Actin.

Figure 10 (A) to (D) illustrates the results of blotting experiments to show that residues Tl 5, S22 and T33 are essential for SpylA degradation.

Figure 11 illustrates a graph showing that aberrant SpylA degradation enhances cell proliferation.

Figure 12 illustrates experiments relating to anchorage independent growth in soft agar.

Figure 13. (A) Sequence composition of five shRNA constructs, each uniquely designed to trigger RNAi-mediated destruction of the Spy 1 A mRNA transcript to variying degrees (1-5). Also included is the coding sequence for the scrambled negative control shRNA construct. (B) Depiction of the mouse SpylA (mSpyl A) protein isoform and its respective amino acid positions that correspond to each shRNA sequence.

Figure 14. (A) Twenty-four hours post-transfection, alterations in cell proliferation were quantified through trypan blue exclusion analysis and cell counting, demonstrating a significant decrease in cell number upon transient SpylA inhibition. (B) SpylA knockdown was confirmed at the protein level through immunoblotting techniques (10% SDS-PAGE), and appeared to downregulate c-Myc in addition to SpylA when normalized to Actin.

Figure 15. Whole mouse dissections displaying fourth inguinal mammary glands following mammary fat pad transplantation of control (left) and SpylA knockdown (right) stable tumor cell lines. Murine subjects were allowed a recuperation period of 3 days (A), or 6 days (B) prior to fresh dissection of No. 4 inguinal mammary glands. Sacrifice of test subjects at Day 3 (A) and Day 6 (B) post-surgery revealed a significant decrease in the rate of mammary tumor formation in those glands exhibiting decreased Spy IA activity. Arrows depict No. 4 mammary glands following differential shRNA treatment.

Detailed Description of the Preferred Embodiments

Cell culture. Human mammary breast cancer cells, MCF7 (ATCC) and human embryonic kidney cells, HEK 293T (293T; ATCC), were maintained in DMEM medium (Sigma) containing 2mM L-glutamine (Sigma), penicillin (Invitrogen), and streptomycin (Invitrogen), and were cultured in a 5% CO ₂ environment. MCF7 cells were supplemented with 10% (vol/vol) fetal calf serum (Sigma) and 293T cells were supplemented with 10% fetal bovine serum.

Plasmids and mutagenesis. The Nedd4-PCEP plasmid (Nedd4), dominant negative Nedd4-PCEP plasmid (Nedd4 ^D ) and empty vector control (PCEP) were provided by Dr. Dale S. Haines (Temple University School of Medicine). HA-Ubiquitin (HA-Ub) was provided by Dr. Sylvain Meloche (Universite de Montreal). A Cyclin B l mutant lacking a portion of the D-box (Cyc BΔD) was provided by Dr. Sylvain Meloche with permission from Dr. Michael Brandeis (The Hebrew University, Israel) and the GFP-Cyclin Bl-CMX vector was provided by Dr. Ed Harlow (Harvard Medical School). Creation of Myc-Spyl A-PCS3 vector was described previously (Porter et al 2002). QuikChange Multi-Site-Directed Mutagenesis (SDM; Stratagene) was used to incorporate new silent sites into the original Spyl-pJT0013 vector (24) in order to facilitate the cloning of truncation mutants A (TMA), B (TMB), C (TMC), G (TMG) and Z (TMZ). A BgIII site was inserted by altering nucleotide 256 from T to C using the primers #A043 5'-

GACGATTTAATTCAAGATCTCTTGTGGATGGACTGCTGC-S' and #A044 5'- GCAGCAGTCCATCCACAAGAGATCTTGAATTAAATCGTC-S' to construct the pRAOl vector. Using the pRAOl plasmid a MIu site was also added by altering nucleotide 175 from C to G using #A004 5'- CAACAAATCTAAACGCGTCAAAGGACCTTGTCTGG-3' and #A005 5'- CCAGAC AAGGTCCTTTGACGCGTTTAGATTTGTTG-S' to make the vector pRA02. The pRS2 vector was constructed from Spyl-pJT0013 by creating an Ndel site just after the stop codon using the primers #A045 5'-

GTCTTGTGTCC ATATGTGTTTTGTGGTGACCC-S ' and #A046 5'- GGGTCACCACAAAACACATATGGACACAAGAC-3'. The pRSl vector was constructed by creating a MIuI site in the Spyl-p JTOO 13 plasmid by altering nucleotide nucleotide 175 from C to G using primers #A004 5'- CAACAAATCTAAACGCGTCAAAGGACCTTGTCTGG-S' and #A005 5'- CCAGACAAGGTCCTITGACGCGTTTAGATTTGTTG-3'. TMA was created by digesting wild-type Spy IA (in pRSl) with Ndel and MIuI in order to remove the first 57 amino acids of the protein. TMB was created by digesting wild-type SpylA (in pRA02) with MIuI and BgIII in order to remove 27 amino acids. TMC was created by digesting wild-type SpylA (in pRAOl) with BgIII and Ncol in order to remove 61 amino acids. TMG was created by digesting wild-type SpylA (in pJT0013) with Ncol and Bbsl in order to remove 94 amino acids. Finally, TMZ was created by digesting wild-type SpylA (in pRSl) with Bbsl and Ndel in order to remove the last 47 amino acids. Gel electrophoresis of these digestions was run on a 1% agarose gel; the desired band was excised and gel-extracted (Bio Basics) for ligation using T4 DNA ligase (Fermentas). For all five truncation mutants, linkers containing a silent restriction site, Pstl, and complementary sticky ends were designed, commercially synthesized (Sigma), annealed and utilized in the ligations. In each case, 20 μL ligation reactions were carried out at 22°C for 2-4 hrs. containing a 1:3 vector to linker ratio. Ligations were transformed into DH5α cells and selected for ampicillin resistance, mini-prepped, and digested with Pstl (Fermentas) to detect the correct ligation. The five SpylA truncation mutants (depicted in FIG. 3A), spanning the length of the gene, were moved from the pJT0013 into pCS3 using EcoRI and Xbal sites flanking the gene. Successful cloning was determined by DNA sequencing (Robarts Sequencing Facility; Univ. of Western Ontario). SDM was also carried out using the PC S 3 vector to generate the Spy IA- Tl 5A, SpylA-T33A and SpylA-S22A mutants. SpylA-T15A was designed using the primers #A151 5' -

GAGACACCACCTACTGTCGCTGTTTATGTAAAATCAG-S' and #A152 5'- CTGATTTT AC AT AAAC AGCGAC AGTAGGTGGTGTCTC-3 ' ; Spy 1 A-T33 A was designed using the primers #A-153 5'-

CAGCCTAAAAAGCCCATTGCACTGAAGCGTCCTATTTG-3' and #A154 5'- CAAATAGGACGCTTCAGTGCAATGGGCTTTTTAGGCTG-S ^• ; Spy IA-S 22A was designed using the primers #A139 5'- GTTTATGTAAAATCAGGGGCCAATAGATCACATCAGC-S ' and #A 140 5 ' - GCTGATGTGATCTATTGGCCCCTGATTTTACATAAAC-S.

Inhibitors and antibodies. The following antibodies were used: Spy IA (NB 100-2521 ; Novus), Nedd4 (abl4592; Abeam), Myc (9E10 and C19; Santa Cruz), HA (Yl 1 and F7; Santa Cruz), Actin (MAB1501R; Chemicon), Cyclin E (551 157; BD Pharmingen). The calpain inhibitor N-Acetyl-L-leucyl-L-leucyl-L- norleucinal N-Acetyl-Leu-Leu-Norleu-al (LLNL), the proteasome inhibitor MGl 32 and nocodazole were all purchased from Sigma.

Transfections. Calcium Phosphate Precipitation transfections were carried out in 293T cells using 10 μg of DNA per 10 cm tissue culture plate. 250 μL CaCl ₂ was incubated with the DNA for 30 sec, 250μL 2x BBS at pH 7.01 was added while vortexing and the solution was incubated for 10 min. The mixture was added slowly to the cells and then incubated in 3% CO ₂ for 12-16 hrs. Media was then changed and plates were returned to 5% CO ₂ for 24 hrs. prior to harvest.

Cell synchronization and flow cytometry. 293T cells were synchronized using double thymidine block. Briefly, cells were cultured in media containing 2 mM thymidine for 16 hrs., followed by release into normal media for 8 hrs. and then a second thymidine block for 14 hrs., and then release into media containing 70 ng nocodazole. MCF7 cells were synchronized by being cultured in a serum-free media for 48 hrs., followed by release into media containing serum and 7Ong nocodazole. 293T and MCF7 cells were trypsinized at specified times, washed twice in PBS, and then either used immediately or fixed and stored at -2O ⁰ C. Fixation was carried out by resuspending cells at 2 x 10 ⁶ cells in 1 niL of PBS, followed by slow addition of an equal amount of 100% ethanol. Within 1 week, fixed cells were pelleted, washed, and resuspended in 300 μL of PBS. Samples were then prepared for flow cytometry by treating with 1 μL of 10 mg/mL stock of DNase free RNase (Sigma) and 50 μL of 500 mg/mL propidium iodide stock solution. Data was collected using a Beckman Coulter FC500 (Biology Dept; U of Windsor) and cell cycle profiles were analyzed using CPX Beckman Coulter FC500 software.

Immunoblotting. Cells were lysed in 0.1% NP-40 lysis buffer (5 mL10% NP-40, 10 mL IM Tris pH 7.5, 5 mL 0.5M EDTA, 10 mL 5M NaCl up to 500 mL RO water) containing protease inhibitors (PMSF 100 μg/mL, aprotinin 5μg/mL, leupeptin 2μg/mL) for 30 min on ice. Bradford Reagent was used to determine the protein concentration following the manufacturer's instructions (Sigma). Aliquots of lysates containing 20-30 μg protein were subjected to electrophoresis on denaturing SDS- 10% polyacrylamide gels and transferred to PVDF-Plus transfer membranes (Osmonics Inc.) for 2 hrs. at 30V using a wet transfer method. Blots were blocked for 2 hrs. in TBST containing 3% non-fat dry milk (blocker) at room temperature. Primary antibodies were reconstituted in blocker and incubated over night at 4 ⁰ C at a 1 : 1000 dilution for all antibodies, and secondary antibodies were used at a 1 : 10,000 dilution in blocker for 1 hr at room temperature. Blots were washed three times with TBST following incubation with both the primary and secondary antibodies. Washes were 6 min each following the primary antibody and 10 min each following the secondary antibody. Chemilumiminescent Peroxidase Substrate was used for visualization following the manufacturer's instructions (Pierce). Chemiluminescence was quantified on an Alpha Innotech HD2 (Fisher) using AlphaEase FC software. Immunoprecipitation reactions were carried out using equal amounts of protein (~200μg/mL) incubated with 2 μg of primary antisera, as indicated, overnight at 4 ⁰ C. This was followed by the addition of protein A-Sepharose (Sigma) and incubated at 4 ⁰ C with gentle rotation for an additional 2 hrs. Complexes were washed extensively with 0.1% NP-40 lysis buffer and resolved by 10% SDS-PAGE.

In vivo labeling. 293T cells were treated with lOμM MG132 and 70 ng nocadozol for 14 hrs. followed by incubation in phosphate-free media for 2 hrs. and then addition of ImCi of [ ³² P] -orthophosphoric acid (GE healthcare) for 4 hrs. Cells were lysed and immunoprecipitated with Myc antisera.

Immunoprecipitations were washed rigorously with TBS and samples were analyzed by 10% SDS page gel. Gels transferred to PVDF membranes were visualized using a Cyclone phosphoimager and quantified using OptiQuant software (Perkin Elmer; Biology Dept.; U of Windsor).

In Vivo Ubiquitination Assays. 293 cells were plated and transfected appropriately in a 100-mm dish. 24 hrs. after transfection cells were treated with 10 μM MG 132 for 14 hrs. Cells were then collected, pelleted by centrifugation, lysed in 200 μl of preboiled lysis buffer [50 mM Tris-HCl (pH 7.5),0.5 mM EDTA, 1% SDS, and 1 mM DTT], and further boiled for an additional 10 min. Lysates were clarified by centrifugation at 13,000 rpm on a microcentrifuge for 10 min. Supernatant was diluted 10 times with 0.5% NP40 buffer and immunoprecipitated with anti-Myc antibody. Immunoprecipitates were washed 3 times and resolved by 10% SDS-PAGE, followed by immunoblotting with anti- HA antibody.

siRNA Knockdown Process. siRNA against Nedd4-1 was synthesized by inserting the oligo 5 'GATGAAGCCACCATGTATA into the pSUPER-basic vector, as previously described . As a control, LacZ siRNA (siCntl) was synthesized and inserted into pSUPER-basic vector as described.293 cells were transfected using 12 μg of either Neddd4 siRNA or siCntl per 100 mm tissue culture plate and total protein was isolated from cell cultures and resolved using 12% SDS polyacrylamide gels as described above.

Tissue Microarray Detection and Quantification of Spyl Protein levels

De-paraffϊnization:

Notice: Before deparaffinization in xylene, tissue (microarray) section slides shall be baked in oven at 6O ⁰ C for 30 minutes on a vertical rack to melt the extra layer of coated paraffin. Trial slides do not have paraffin coating so it can be deparaffinized without baking. All of our tissue (microarray) section slides, unless otherwise specified, were baked at 60 ⁰ C for two hours after sectioning and are stored at 4 ⁰ C.

1. Immerse slide in xylene for 10 minutes. Repeat once in new xylene for 10 minutes.

2. Immerse array slide in 100% ethanol for 5 minutes.

3. Immerse in 95% ethanol for 5 minutes.

4. Immerse in 70% ethanol for 5 minutes.

5. Rinse for 5 minutes in water or PBS buffer.

Immunostaining using fluorescent probes

1. Deparaffinize and dry array slide as referred to in protocol of deparaffinization.

2. Rinse array slide twice with PBST for 5 min each in a Coplin jar.

3. Antigen retrieval (formalin-fixed, paraffin-embedded tissue sections). Boiling Bath. Heat the buffer (ImM EDTA, pH 8.0 or 0.01M sodium citrate buffer, pH 6.0) to about 95 ⁰ C, and then put array slides in the buffer for 10-15 min. Do NOT let the medium boil when you have array slide in. Avoid the slide drying during the procedure.

4. Rinse array slide in PBST for 5 min.

5. Apply the blocking antibody (normal goat serum- 150ul/10ml), incubate for 40 min at room temperature, and throw off residual fluid (don't wash.).

6. Apply the primary antibody 60 min.at RT (Spyl Novus 1 :50) usually 200ul volume to cover all the cores.

7. Rinse array slide twice for 5 min each in a Coplin jar on the orbital rotator

8. Incubate array slide with a fluorophore-conjugated secondary antibody at 20~37°C (in a humidity chamber) for 20 min. (Alexa488 1 : 1200)

9. Rinse array slide twice in PBST for 5min each in a Coplin jar on the orbital rotator.

10. Incubate array slide with TOTO-3 nuclear stain (0.75ul: 1000) in PBST at RT 30 min.

11. Rinse array slide 3 times in PBST for 5min each in a Coplin jar on the orbital rotator.

12. Dehydration and transparency of array slide.

1. Immerse in 70% ethanol for 5 minutes.

2. Immerse in 95% ethanol for 5 minutes.

3. Immerse array slide in 100% ethanol for 5 minutes.

4. Immerse slide in xylene for 10 minutes. Repeat once in new xylene for 10 minutes 13. Mount array slides with the Vectashield mounting media and cover with a square or rectangulat cover slip no.l .

14. The TMA slide is being placed in the slide scanner. The edges and surface of the cover slip have to be clean and dry in order to place the slide in the slide scanner. The fluorescent signal for the secondary antibody fluorophore and nuclear stain is detected and quantified by ScanArray Express software (Perkin Elmer Inc.)

15. The mean the fluorophore signal intensity values are normalized to the mean of the nuclear stain signal.

The results are shown in Figure 6.

To correlate the expression levels of Spy 1 with the type and grade of brain tumour we used tissue microarrays (TMAs) consisting of 1 03 individual patient cores from malignant and benign human brain tumour samples and normal matching brain tissues. The TMAs were probed with anti-Spy 1 primary antibody followed by staining with Alexa-488 conjugated secondary antibody. ToTo-3 stain was used as nuclear control. The fluorescent signal was detected and quantified by ScanArray Express (Perkin Elmer Inc.) The Spy l signal intensity was normalized to nuclear stain signal. We found that Spy l expression levels were highly elevated in tumour tissues comparing to normal brain tissue (Fig 6A). Moreover the proportion of Spy 1 positive cells increased with tumour grade for both oligodendro- and astrocytic gliomas and correlated with higher expression in malignant tumours (Fig 6A ₅ B)-

Tissue protein extraction and Western Blot protocol for SpylA protein levels:

1. Tissue samples were stored at -80'C. 2. Each sample was allowed to thaw on ice for lOmin.

3. Tissue lysis buffer:

• 5OmM Tris-HCl, pH 7.4;

• 1% NP-4O; • 0.25% sodium deoxycholate;

• 15OmM NaCl;

• ImM EGTA;

• ImM PMSF;

• lug/ml each aprotinin, leupeptin, • Antifoam 50ul

Make sure the buffer is cold!

4. Tissues were homogenized in Eppendorf tubes using plastic pestles 20min and incubated another 45 min on the ice.

5. The samples were centrifuged at 13000 rpm for 15 min and the supernatant containing the protein extract was stored at -20'C.

6. The protein concentration was estimated using Bradford assay.

7. Samples for the SDS-PAGE analysis were prepared using 4x sample/loading buffer containing SDS and glycerol.

8. 10% Polyacrylamide gel was prepared according to standard protocol.

9. Prior to loading samples were boiled for 5min.

10. Gel ran at 110V 25mA for 3.5h in a Ix SDS running buffer (25mM Tris- HCl; 20OmM Glycine; 0.1% SDS)

1 1. PVDF membrane was activated in methanol and soaked in transfer buffer for 3 min. 12. The gel was taken out from the cast and soaked in the transfer buffer for lOmin.

13. "Western Blot sandwich" was assembled and placed in the transferring chamber.

14. The transfer ran at 30 V for 2hr.

15. The membrane was blocked in 2.5% milk in TBST for lhr with shaking.

16. The membrane was incubated o/n at 4'C on a shaker with the proper primary antibody (Spyl Novus 3ul/ml; p27 Calbiochem 1 : 1000; Actin Santa Cruz 1 : 1000) diluted in 2.5% milk.

17. The membrane was washed 3x 15 min in TBST and incubated with the proper secondary antibody 1 : 10 000 diluted in 2.5% milk for lhr at RT on a shaker.

18. The was washed 3x 5 min with TBST.

19. Detection by ECL. The blot was exposed using Alphalnnotech Imager and software.

The results are shown in Figure 7.

We obtained glioblastoma (Fig 7C), oligodendroglioma (Fig 7B), oligoastrocytoma (Fig 7A) and pair matched normal tissues samples from the Ontario Tumour Tissue Bank. We tested samples obtained from biopsies taken from the tumour center (i), peritumour (ii-iv) samples and normal brain tissue (v). Total protein was extracted from frozen tissues and subjected to western blot analysis. Samples were probed using Spyl , actin, and p27 antibodies. Spyl levels were found to be elevated in the tumour center as compared to the normal tissue in 9 of 12 patient samples.

Immunohistochemistry detection of SpylA protein levels 1. Sections placed on slides were deparaffϊnized in Xylene (2x5min in a Coplinjar)

2. Rehydrated in 100% ethanol ( 1 x5min)

3. Blocked in 3% H ₂ O ₂ in 100% methanol ( 1 x20min)

4. Rehydration continued

• 100% ethanol ( 1 x5min)

• 95% ethanol (Ix5min)

• 70% ethanol (Ix5min)

• DI water (Ix5min) • IxPBS (Ix5min)

5. Antigen retrieval. Citrate buffer pH 6.0; 600ml in 2L beaker

Slides were placed in a wheaton glass slide holder and subjected to 4 rounds x 5 min in 100OW microwave on high. Lost volume of watr was replaced with dd water. The slides were cooled for 20min and washed in water 4x2min and Ix2min in IxPBS.

6. The sections were blocked using 10% normal goat serum in PBST (0.1% Tween) minimum Ihr. The blocker was drained not washed.

7. The sections were incubated with the primary antibody (1 : 100) for Ihr.

8. The antibody was washed away with PBST 3x5min

9. The secondary Ab was applied (1 :200) goat anti-mouse biotin conjugated and incubated for 45' at RT.

10. The sections were washed 3x5min.

11. ABC solution was applied for 45 ' at RT and washed 2xPBST. 12. The sections were washed IxPBS

13. The sections were treated with the DAB solution and observed under the dissection microscope till they reached proper staining level.

14. The DAB was washed with IxPBS for 5min.

15. The sections were counterstained with hematoxylin for 40" and washed in water.

16. The sections were dehydrated

• 70% ethanol Ix5min

• 95% ethanol Ix5min • 100% ethanol Ix5min

• 100% ethanol Ix5min

• Xylene 3x 5min

17. The slides were mounted with Permount followed by cover slip.

The results are shown in Figure 8.

Generation of deletion mutant A and the non-degradable point mutations.

DMA was created by digesting wild-type Spyl A with Ndel and MIuI in order to remove the first 57 amino acids of the protein. Gel electrophoresis of these digestions was run on a 1% agarose gel; the desired band was excised and gel-extracted (Bio Basics) for ligation using T4 DNA ligase (Fermentas). Linkers containing a silent restriction site, Pstl, and complementary sticky ends were designed, commercially synthesized (Sigma), annealed and utilized in the ligations. In each case, 20 μL ligation reactions were carried out at 22°C for 2-4 hrs. containing a 1 :3 vector to linker ratio. Ligations were transformed into DH5α cells and selected for ampicillin resistance, mini-prepped, and digested with Pstl (Fermentas) to detect the correct ligation. The five Spyl A deletion mutants (depicted in Fig. 9), spanning the length of the gene, were moved from the pJT0013 into pCS3 using EcoRI and Xbal sites flanking the gene.

SDM was also carried out using the PCS3 vector to generate the Spy 1 A-

T15A, SpylA-T33A, SpylA-S22A and SpylA-S247A mutants. SpylA-T15A was designed using the primers #A151 5'-

GAGACACCACCTACTGTCGCTGTTTATGTAAAATCAG-3' and #A152 5'-

CTGATTTTACATAAACAGCGACAGTAGGTGGTGTCTC-3'; Spyl A-T33A was designed using the primers #A-153 5'-

AGCCTAAAAAGCCCATTGCACTGAAGCGTCCTATTTG-3' and #A1545'- CAAATAGGACGCTTCAGTGCAATGGGCTTTTTAGGCTG-S'; Spy1A-

S22A was designed using the primers #A139 5'-

GTTTATGTAAAATCAGGGGCCAA TAGATCAC ATCAGC-3' and #A140

5 ^" -CTGATGTGATCTATTGGCCCCTGATTTTACATAAAC-S; SpylA-S247A was designed using the primers #A143 5'- GGATTGTCTTCATCATCAGCGTTATCCAGTCATACTGCAGGGGTG-S ' and #A 144 5 '-

CACCCCTGCAGTATGACTGGATAACGCTGATGATGAAGACAATCC-S ' .

Successful cloning in all cases was determined by DNA sequencing (Robarts

Sequencing Facility; Univ. of Western Ontario).

The results are shown in Figure 9.

SpylA degradation depends on phosphorylation within the N- terminal region. Using a panel of SpylA deletion mutants (Fig. 9A), we began to narrow down the region within the SpylA protein that was necessary for degradation. We first determined whether deletion of any of the regions of SpylA would result in stabilization of the protein. 293 cells were transfected with wild- type SpylA or deleted versions of the SpylA protein, DMA-DMZ. Cells were synchronized at G ₂ /M and levels of SpylA were monitored by immunoblotting (Fig. 9B; upper panel). All deletion mutants of SpylA were degraded by G ₂ /M phase with the exception of the mutant lacking the first 57 amino acids (DMA). Asynchronous cells demonstrate that all deletion mutants were expressed (Fig. 9B; lower panel). Collectively, these data demonstrate that the N-terminal region of Spy IA is essential to mediate degradation of the protein and that unlike the Xenopus homolog of Spy 1 the C-terminal region is dispensable for degradation.

Residues T15, S22, and T33 are essential for SpylA degradation. We have demonstrated that the N-terminal region of SpylA is essential for mediating degradation. Hence, we focused on elucidating sites within this region that may target the protein for degradation. Utilizing the NetPhos 2.0 Server tool residues Tl 5, S22, and T33 were isolated as potential phosphorylation sites. Site-directed mutagenesis was performed to alter SpylA residues T15, S22, and T33 to non- phosphorylatable alanines. Additionally we generated a similar mutation at S247 in the C-terminal region to serve as a control. 293 cells were transfected with the relevant constructs prior to synchronization at G ₂ /M. Surprisingly, mutation of all of Tl 5, S22, and T33 to a non-phosphorylatable alanine prevented degradation and ubiquitination of SpylA (Figs. 1OA & B). Blotting asynchronous cell populations revealed that protein expression was not affected (Fig. 1 OA; right panel). This suggests that phosphorylation, or maintenance of charge of all three of T15, S22, and T33 is essential in regulating the turnover of SpylA. To further assess the effect of these mutations on SpylA degradation, 293 cells were transfected and then treated with 50 μg/ml cyclohexamide 16hrs. post- transfection. Immunoblotting for SpylA showed that cells transfected with the mutants have stabilized SpylA levels (Fig. 10C). Quantifying 3 separate experiments demonstrate that indeed all 3 mutations significantly enhance the stability of SpylA protein (Fig. 1OC; right hand panel). To assess whether these sites are phosphorylated in vivo a triple mutant (Spy IA-TST) was created where all 3 elucidated sites were mutated to a nonphosphorylatable alanine (Tl 5A, S22A and T33A). Phosphorylation of Spyl A-TST at G2/M was compared to that of wt-SpylA using an orthophosphate labeling experiment. A significant decrease in phosphorylation was observed with the triple mutant (Fig. 10D), demonstrating that SpylA is phosphorylated at residues T15, S22 and T33 during G2 phase of the cell cycle.

The results are shown in Figure 10.

Aberrant SpylA degradation enhances cell proliferation. To test the effects of ablating SpylA degradation on cell proliferation live and dead cell populations were monitored by trypan blue analysis. SpylA and mutant constructs significantly enhance cell proliferation as compared to mock with p values of 0.01 for mock: WT, 0.001 for mock: Spy IA-T 15 A, 0.0004 for mock: Spy IA- T33A, and 0.001 for mock:Spyl A-S22A. There was no statistical change in the number of dead cells from one transfection to another (Fig. 1 1; grey bars). Interestingly, Spyl degradation mutants statistically enhanced proliferation over SpylA alone by 20-60% (Fig. 11; black columns), p-values for these comparisons were 0.009 for WT:SpylA-T15A, 0.002 for WT:SpylA-T33A and 0.03 for WT:SpylA-S22A.

The results are shown in Figure 1 1.

Oncogenic effects of TST

Anchorage Independent Growth in Soft Agar. It was examined whether non-degradable Spyl is able to acquire a transformed phenotype through a soft agar assay testing for anchorage independence. The assay was done in triplicates, including 4 transfections (WT, TST, PCS3 and RASV12). The assay was conducted 3 times as three separate experiments. Overall, the assay was incubated for 10-14 days before the colonies were photographed.

Consistent with anchorage independent growth and invasive phenotype in vitro, RASVl 2 yielded numerous colonies grown in soft agar as it was expected. (Figure 12A, 12B and 12C)Surprisingly, when TST was plated and allowed to grow in soft agar, numerous colonies were observed as well. (Figure 12A, 12B and 12C) However when WT Spyl and PCS3 were grown in soft agar, no apparent colonies formed and the few colonies that did form for WT Spyl were not the same size or magnitude as the RASV12 or TST formed colonies. (Figure 12B).

The results are shown in Figure 12.

Screening for TST in different cancerous cell lines. To examine if these mutations are existing in some cancerous cell lines fife cell lines were utilized;

HCT p21-/-, HCT p53 -/-, MCF7, HTB 231 and HTB 126. Using sequencing primers A455 and 457 we were able to PCR DMA and purify it using Biobasic

PCR purification kit and sequence at Robart sequencing facility at University of

Western Ontario. The sequencing results showed that amino acids from 1-61 (DMA) are not mutated in all the cell lines being tested.

MRHNQMCCETPPTVTVYVKSGSNRSHQPKKPITLKRPICKDNWQAFEKNT

MRHNQMCCETPPTVTVYVKSGSNRSHQPKKPITLKRPICKDNWQAFEKNT 10 20 30 40 50

160 170 HNNNKSKRPKGPC

HNNNKSKRPKGPC

60

Tool Development for shRNA Knockdown of Spyl Protein Levels.

Available online as free software (http://www.oligoengine.com), the program OligoEngine Workstation 2 was utilized to design five novel shRNA constructs directed against unique regions of the mouse Spyl A isoform, in addition to a scrambled shRNA construct chosen for its inability to recognize and target Spyl A for RNAi-mediated degradation. Constructs flanked by Hpal and Xhol restriction sites were synthesized by Sigma-Genosys Canada (Sigma-Aldrich, Ontario) and subsequently subcloned into pLB (Addgene, MA, USA), a lentiviral vector that utilizes the mouse U6 promoter to dually express shRNA and green fluorescent protein (GFP) in mammalian cell systems. Thus, GFP fluorescence served as a positive indicator for induction of RNAi.

The results are shown in Figure 13.

Transient SpylA Knockdown in Primary c-Myc Overexpressing

Tumor Cell Lines. The MMTV-Myc mouse model (MMHCC, NCI), well documented for its ability to form aggressive mammary tumors, was utilized to derive a previously uncharacterized tumor cell line engineered to overexpress the proto-oncogene c-Myc. Polyethylenimine (PEI) was utilized to transfect immortalized primary cell cultures with control or SpylA knockdown vectors.

Stable Cell Line Production & Mammary Fat Pad Transplantation: In vivo Knockdown of SpylA. PEI -mediated transfection of the HEK-293T producer cell lines was utilized to introduce control or Spyl A-shRNA vectors to a series of pre-optimized packaging vectors that code for the necessary viral components required to form lentiviral particles upon transduction of mammalian cell lines. Following production of infectious lentivirus, control and SpylA knockdown particles were each concentrated and titered as previously outlined in WeIm, B.E., et al. (2008), Cell Stem Cell 2(1): 90-102, proceeded by infection of primary tumor cell lines with respective viral concoctions. Subsequently, stable cell lines were passaged three times, after which they were utilized to perform mammary fat pad transplantations in 28-day old FVB female mice. In summary, number four glands were cauterized to prevent endogenous stem cell populations from colonizing the mammary fat pad, which in turn served as an environmental substrate to support transplanted stable cells of either type. Left cleared fat pads were injected with control shRNA stable cells, whereas right cleared fat pads were injected with SpylA shRNA stable cells (Fig.15).

SpylA protein degradation occurs in a cell cycle dependent fashion. It is known that spy IA is a low copy number gene which is constitutively expressed in most human tissues and that the mRNA is specifically up-regulated in the Gi phase of the cell cycle (5, 24) unpublished data). Herein we wished to determine whether levels of the protein are regulated in a cell cycle dependent fashion, or whether levels vary independently of cell cycle stage. In order to address this question, MCF7 and 293T cells were blocked at Gi phase of the cell cycle using serum starvation (SS) or a double thymidine block (TB), respectively, followed by release into nocadozol containing media. A G ₂ population was collected at 16 hrs following release for both cell lines (nocadozole treatment; NT). Flow cytometry analysis was used to confirm the cell cycle stage for cells harvested during the block (Gi) or following release (G ₂ ) (FIG. IA and C; upper panels). Asynchronous populations were also used as a control (Cntl). Percentages of cells in each phase of the cell cycle, as determined by flow cytometry analysis software, are shown in graphic form (FIG. IA and C; lower panels). Immunoblotting of cell lysates from different stages o the cell cycle showed that Spy 1 A protein levels are greatly decreased at the G ₂ /M border (FIG. IB and D; upper panels). Cyclin E was assessed in order to ensure that cells were in the proper stage of the cell cycle (FIG. IB; middle panel), and Actin was used as the loading control (FIG. IB and D; lower panels). This demonstrates that SpylA, like many important cell cycle proteins, is tightly regulated in a cell cycle dependent fashion.

To further narrow down where in the cell cycle SpylA was being degraded, we utilized a non-degradable Cyclin Bl mutant (CycBΔD). It is well established that Cyclin Bl must be degraded in order for cells to successfully complete the metaphase-to-anaphase transition (34). The CycBΔD contains a 9 residue motif mutation within the destruction box, thus disrupting the region responsible for the association between Cyclin Bl and the ubiquitin ligase, APC (35). Hence, the CycBΔD mutant of Cyclin Bl arrests cells at the metaphase-to- anaphase transition. 293T cells were transfected with wild-type Cyclin Bl- PEGFP cDNA (CycBl), CycBΔD (CycB-D) or an empty PEGFP vector (GFP). Cell lysates from the transfections or an untransfected control (Cntl) were analyzed by immunoblotting. Cells transfected with CycBl and GFP contained similar levels of SpylA as non-transfected cell lysates (FIG. IE; lanes 1, 2, and 3). However, there was no SpylA protein detected in the lysates from cells overexpressing the CycBΔD mutation (FlG. IE; upper panel; lane 4). Actin was used as a loading control (FIG. IE; lower panel). This data demonstrates that the degradation of SpylA takes place prior to the anaphase transition during mitosis.

SpylA degradation is proteasome dependent. After determining the timing of SpylA degradation during cell cycle progression, we set out to investigate the mechanism by which this occurs. Gutierrez et al. have reported that X-Spyl is being processed and degraded via the ubiquitin proteolytic pathway, and as such, this was the pathway we began to investigate. 293T cells were transiently transfected with HA-tagged ubiquitin (Ha-Ub) and Myc -tagged SpylA (Myc-SpylA), followed by immunoprecipitation with anti-Myc antibody (FIG. 2A; lower panel). Immunoblotting with anti-HA antibody revealed that SpylA was labeled with HA-ubiquitin in vivo (FIG. 2A; upper panel). To verify the involvement of the proteasome machinery in SpylA turnover, we studied

293T cells in the presence or absence of the proteasome inhibitor MG132 or the calpain inhibitor LLNL. SpylA protein levels were significantly elevated in the presence of the proteasome inhibitor MG132 (FIG. 2B; upper panel; lane 4) but not in the presence of the calpain inhibitor or the vehicle controls (FIG. 2B; upper panel; lanes 1-3). Actin was used as a loading control (FIG. 2B; lower panel). This data implicates that SpylA protein degradation is proteasome dependent.

SpylA degradation depends on phosphorylation within the N- terminal region. Using a panel of SpylA truncation mutants (depicted in FIG. 3A), we began to narrow down the region within the SpylA protein that was necessary for degradation. We first determined whether deletion of any of the regions of SpylA would result in stabilization of the protein. 293 T cells were transfected with wild-type SpylA (wt), empty PCS3 vector (-ve), or truncated versions of the SpylA protein, TMA-TMZ (A-Z). Cells were synchronized at G ₂ /M with nocadozole, and levels of SpylA were monitored by immunoblotting (FIG. 3B; upper panel); Actin was used as a loading control (FIG. 3B; lower panel). This figure demonstrates that all truncation mutants of Spy IA were degraded in the G ₂ /M phase with the exception of TMA (FIG. 3B; upper panel; lane 3). This suggests that the N-terminal region of SpylA is essential to mediate the degradation. Furthermore, the molecular weight of the resolved SpylA was 53 kDa, which is the size of the wild-type tagged SpylA protein. This demonstrates that either the SpylA protein is not processed prior to degradation, as is the case with X-Spyl, or that the N-terminal region of SpylA is required for both processing and degradation of the SpylA protein.

Phosphorylation is often the key event regulating recognition of the substrate protein by the E3 (4). To determine whether TMA was altering the phosphorylation status of SpylA during G ₂ , orthophosphate labeling was performed using the panel of SpylA truncation mutants. A significant decrease in the incorporation of orthophosphate was observed for TMA (FIG. 3C; upper panel; lane 3), surprisingly however no substantial decrease in phosphorylation was observed for any of the other truncation mutants (FIG. 3C; upper panel; lanes 4-7). From this information, we conclude that there is at least one phosphorylation site present within the N-terminal region of SpylA that may play a significant role in regulating SpylA stability. We have presented an alignment of the SpylA and X-Spyl proteins showing the regions known to be essential for processing and degradation of the X-Spyl protein (FIG. 3D). Notably the majority of regions associated with X-Spyl degradation reside within the C- terminal region, a region which was dispensable for mediating SpylA degradation (FIG. 3B).

Furthermore, the sites required for phosphorylation of X-Spyl to initiate processing, T8 and S 12, are not conserved in the mammalian homologue (FIG. 3D; bold, +). Additionally, the phosphorylation sites known to be essential for mediating binding of the SCF ^βTrCP ligase (S233 and S237) are also not completely conserved in SpylA. The ubiquitin ligase Siah-2, which regulates the final degradation process in X-Spyl, depends on S243 phosphorylation by CDK2 for binding. This recognition site requires the adjacent proline at residue 244, which is not conserved in the mammalian homologue (FIG. 3D, bold **). This supports that degradation of SpylA in the somatic cell likely occurs via a different mechanism than that of the Xenopus homologue during oocyte maturation.

Nedd4 is the E3 ligase responsible for SpylA degradation. There are many different E3 ubiquitin ligase enzymes that are able to function in the ubiquitination pathway, therefore we set out to determine which one played a role in the degradation of SpylA. A protein blast for the N-terminal region of SpylA revealed a weak potential interaction region for WW domain containing proteins, PPxxxxY (FIG. 3D; grey shading). It is known that the WW domain-containing ligase, Nedd4 (product of neuronal precursor cell-expressed developmentally down-regulated gene 4), while preferring the canonical PPxY sequence, also binds to a variety of proline rich regions with phosphorylated threonine or serine residues to trigger ubiquitination and subsequent degradation (12, 30, 31). Due to this we investigated the potential role of Nedd4 in SpylA degradation. Nedd4 is a family of conserved E3 ubiquitin ligases required for ubiquitination of a large number of target proteins (30). Isoforms of Nedd4 exist in the simplest yeast cells to the most complex mammals, and have been shown to assist with regulating developmentally-expressed proteins (15, 22). Co-immunoprecipitation assays in cells overexpressing exogenous Nedd4 as well as Myc-tagged SpylA (Myc-SpylA) demonstrate that Nedd4 interacts with SpylA in vivo (FIG. 4A; upper panel; lane 3). To further investigate whether Nedd4 is functioning as an ubiquitin ligase for SpylA, we repeated the co-immunoprecipitation experiment using overexpression of wild-type Nedd4 (Nedd4) or dominant negative Nedd4 (Nedd4 ^DN ) in the presence of HA-tagged Ubiquitin (Ha-Ub). Immunoblotting for HA-tagged Ubiquitin, followed by quantification, revealed that SpylA incorporated 41 % HA-Ub in the presence of Nedd4, and that this was significantly decreased (by 18%) in the presence of Nedd4 ^DN (FIG. 4B; upper panel; lane 3). Quantification is depicted in FIG. 4B; right graph. To further assess the effect of Nedd4 on endogenous SpylA, Nedd4 was transfected into 293T cells in the presence and absence of the proteasome inhibitor MG132, and endogenous levels of SpylA were measured. SpylA protein levels were decreased by 20% when Nedd4 was transfected in the absence of MGl 32 (FIG. 4C; upper panel; lane 3) as compared to when MG132 was present (FIG. 4C; upper panel; lane T). Densitometry was performed and equalized for protein loading as determined by Actin (FIG. 4C; lower panel; right graph). Collectively, this data demonstrates that SpylA binds to the E3 Nedd4 and that Nedd4 is capable of enhancing ubiquitination of SpylA and promoting the degradation of SpylA.

SpylA phosphorylation at T15, S22, and T33 regulates SpylA degradation. Cell cycle regulatory proteins which are targeted to the UPS rely on signal transduction mechanisms to control the timing of this essential event. We have demonstrated that the N-terminal region of SpylA (depicted in FIG. 3D) is essential for mediating degradation. Hence, we focused on elucidating potential phosphorylation sites within the N-terminal region of SpylA that may target the protein for degradation. Utilizing the NetPhos 2.0 Server tool, which predicts potential serine, threonine, and tyrosine phosphorylation sites, residues T15, S22, and T33 were shown to have a high probability of being potential phosphorylation sites (3). Site-directed mutagenesis was subsequently performed to alter SpylA residues Tl 5, S22, and T33 to non-phosphorylatable alanines (Tl 5A, S22A, and T33A respectively). 293T cells were then transfected with wild-type SpylA (wt), SpylA-T15A (T15A), SpylA-S22A (S22A), and SpylA- T33A (T33A) prior to synchronization at G ₂ /M. Surprisingly, mutation of any of the three sites Tl 5, S22, and T33 to a non-phosphorylatable alanine prevented degradation of SpylA (FIG. 5A; left, upper panel; lanes 1-3). Blotting asynchronous cell populations revealed that protein expression was not affected (FIG. 5A; right, upper panel), Actin blotting determined even loading (FIG. 5A; left & right lower panels). This suggests that phosphorylation at all three of Tl 5, S22, and T33 is an essential event in regulating the turnover of SpylA. Cells transfected with wild-type SpylA (wt) and each of the mutants (S22A, T33A, T15A) were also subjected to a number of other experiments. In the presence of MG 132, to prevent degradation, cell lysates were immunoprecipitated with c- Myc antibody and immunoblotted for HA (Ha-Ub-Spyl A; FIG. 5B; upper panel) and Myc (FIG. 5B; lower panel). These results demonstrate that wild-type SpylA is ubiquitinated, however all of the three mutations had no detectable incorporation of Ha-Ub (FIG. 5B; upper panel; lane 4 vs. lanes 1-3). Similarly, this membrane was immunoblotted for Nedd4 (FIG. 5B; middle panel). This panel demonstrates that Nedd4 immunoprecipitates with wild-type SpylA and SpylA-T33A (FIG. 5B; middle panel; lanes 4 and 2), however the SpylA-S22A and SpylA-T15A mutants no longer demonstrate binding to Nedd4 (FIG. 5B; middle panel; lanes 1 and 3). These data demonstrate that S22 and Tl 5 on SpylA are essential phosphorylation sites to mediate Nedd4 binding. This further implies that loss of ubiquitination seen with the SpylA-T33A mutant (FIG. 5B; upper panel; lane 2) might represent the phosphorylation site which mediates ubiquitination. To test the effects of ablating SpylA degradation on cell growth characteristics, wild-type SpylA (WT) and the mutants were transfected into 293T cells, and cell proliferation and flow cytometry were carried out. FIG. 5C depicts the results of 3 separate transfections of each cDNA counted in triplicate. Wild-type SpylA and mutant constructs were shown to significantly enhance cell proliferation over mock with p values of 0.01 for mock: WT, 0.001 for mock:SpylA-T15A, 0.0004 for mock:SpylA-T33A, and 0.001 for mock: Spy IA- S 22A (these stats are not reflected in FIG. 5B). Preventing the degradation of SpylA through the alteration of Tl 5, T33, S33 to non-phosphorylatable alanines statistically enhanced proliferation over SpylA alone by 20-60% (FIG. 5C; black columns), p-values for these comparisons were 0.009 for WT:SpylA-T15A, 0.002 for WT:SpylA-T33A and 0.03 for WT:Spyl A-S22A; these p-values are reflected in FIG. 5C. There was no statistical change in the number of dead cells from one transfection to another (FIG. 5C; grey bars). Flow cytometry analysis was performed using cells from FIG. 5A. Profiles were collected for WT or mutant transfected cells in either an asynchronous population, to determine whether preventing the degradation of SpylA altered the normal cell cycle profile, or in nocodazole treated population, to determine if SpylA overexpression could override a G ₂ /M arrest (FIG. 5D). WT and mutant SpylA constructs demonstrated very similar cell cycle profiles, demonstrating that preventing the degradation of SpylA in this cell type did not trigger any normal cellular responses to prevent cell cycle progression. Collectively, this data demonstrates that residues Tl 5, S22, and T33 within the N-terminal region of SpylA are important phosphorylation sites for mediating the degradation of the protein; Tl 5 and S22 appear to be essential sites mediating the binding of Nedd4, and T33 may be a potential phosphorylation site to trigger ubiquitination events. Furthermore we demonstrate that preventing the degradation of SpylA in the 293T cell line does not trigger cell cycle arrest or prevent nocodazol induced G ₂ /M arrest,however it does result in enhanced cell proliferation.

Importance of SpylA Degradation in Cell Cycle Regulation. Tight regulation over the protein levels of cyclins is known to be one essential mechanism by which the cell ensures the proper timing of cell cycle events. This regulation is also accomplished through the regulated activity of the CDKs (23). More recently it has come to light that CDKs can also be activated by members of the Speedy/RTNGO family. These proteins lack any sequence homology with cyclins however our data demonstrates that, like the cyclins, SpylA is tightly regulated at the protein level through the cell cycle. It has been demonstrated that X-Spyl can induce cleavage arrest in embryos when overexpressed post meiosis (10); this suggests that, like the cyclins, regulation of X-Spyl levels is necessary to maintain normal procession through the cell cycle. Our data in 293T cells demonstrates that preventing the degradation of SpylA did not trigger a cell cycle arrest of the somatic cell cycle. This may reflect a bone fide species or cell type difference in regulation of this protein, whereby SpylA is not functioning like a core cyclin in mediating the somatic cell cycle. However, this requires further experimentation in non-immortalized cells. The significance of Speedy/RINGO proteins in the cell cycle is irrefutable, and their tight control is absolutely essential for normal progression; indeed, deregulated levels of Speedy/RINGO proteins and their binding partners are found in many cancer types (19, 36). Therefore, our data demonstrates that preventing the degradation of Spy IA enhances cell proliferation, further supporting a potential role for SpylA in tumorigenesis.

The SpylA-Nedd4 Interaction. Herein, we demonstrate a novel interaction between SpylA and the E3 ligase Nedd4 which mediates the degradation of SpylA. The domain structure of Nedd4 family members are very similar and contain a series of typically two to four WW domains which function as recognition sites for specific substrates or adaptor proteins (1, 15). The WW domains of Nedd4 preferentially recognize PPxY motifs in their substrates (Murillas, 2001). The N-terminal region of SpylA lacks this consensus site; however SpylA does contain a potential PpxxxxY site and it is known that Nedd4 can also interact with phosphorylated threonine or serine residues to trigger ubiquitination and subsequent degradation (30) (31). Following mutagenesis of 3 potential phosphorylation sites within the N-terminal region of SpylA, we have determined that phosphorylation in the region of amino acids 15-33 is generally important for SpylA degradation. Our data has demonstrated that phosphorylation at Tl 5 is particularly important for mediating degradation: Tl 5 is completely conserved among the mammalian SpylA homologues, and is preceded by a highly conserved proline rich region (PPTV). Potential phosphorylation sites at position 15, 22 and 33, or amino acids which mimic phosphorylation are also found among other members of the Speedy/RINGO family in mammals: Spyl/RINGO-B, which is only found in the testes, has a conserved S22 site and contains a glutamic acid at position 15 which may mimic phosphorylation, however it lacks flanking prolines; Spyl/RINGO-C, which is found in the liver, kidney, bone marrow, placenta and testes has a conserved serine at position 15 with two prolines at positions 1 1 and 13, and a conserved T33; whether these sites are involved in proteolysis of other Spyl/RINGO family members remains to be determined.

Furthermore, the Nedd4 family consists of nine members, all containing WW domains. We know from overexpression assays using Nedd4-1 cDNA that this member of the Nedd4 family is capable of interacting with Spy IA and promoting the ubiquitination and degradation; however, whether other members of the Nedd4 family are also capable of regulating the degradation of Spy IA is currently not known. Assays using a dominant negative form of Nedd4-1 did not completely abolish ubiquitination of Spy IA in vivo, however this may reflect an issue of transiently transfecting the dominant negative vector. Future studies into the sufficiency of Nedd4-1 to regulate Spy IA degradation is required.

SpylA-Nedd4 Interaction in Cancer. From the current catalogue of known Nedd4 substrates it appears that Nedd4 can act as both a proto-oncogene, as well as a tumor suppressor under different circumstances. For example, Nedd4 has been shown to mediate the degradation of the vascular endothelial growth factor receptor 2 (VEGF-R2) (21). VEGF-R2 is a positive regulator of cell proliferation, migration, and angiogenesis (7), and it is known to be up- regulated in colon (9), brain (26), and breast cancer (29). In addition, Nedd4 has been shown to lead to the down-regulation of the insulin-like growth factor 1 receptor (IGF-IR) (32), which has been implicated in both the initiation and development of many human cancers types (20). Our data provides further evidence that Nedd4 can function like a tumor suppressor to regulate the levels of proteins stimulating cell growth mechanisms. Conversely, Nedd4, or Nedd4 family members, have been shown to regulate the degradation and function of important tumor suppressor genes such as the phosphatase and tensin homolog 1 (PTEN), p53 and the p53 family member, p73 (17, 33)(28).

Taken together, these conflicting findings underscore the critical importance of resolving the factors that mediate Nedd4 substrate specificity. In addition to understanding the regulatory factors mediating Nedd4 interactions and regulating Nedd4 expression itself, it is also important to resolve the subcellular localization of Nedd4 and its specific substrates. Although primarily cytoplasmic, Nedd4 is known to enter the nucleus, within which it continues its role as a ligase (11). Spy IA, on the other hand, is primarily nuclear however unpublished data has demonstrated that SpylA also shuttles under different circumstances. Furthermore, resolving the expression levels of Nedctø and Nedd4 family members in specific cell types throughout development may resolve apparent conflicting data with regard to the role of Nedd4 mediated actions on cell proliferation. Therefore, in addition to understanding the regulation of temporal control of Nedd4, it is important to also study the spatial subcellular localization of these interactions through real time. Future studies with regard to the regulation of Nedd4 may aid in understanding how Spy IA participates in regulating both normal and abnormal development.

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