METHODS AND COMPOSITIONS RELATING TO PBKl

[0001] This application claims the benefit of priority from United States provisional application 60/939,462, filed May 22, 2007, the entire contents of which is hereby incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING APPENDIX

[0002] Amino acid and nucleic acid sequences are shown in appended pages marked "Appendix" and are also being submitted on the accompanying compact disc; said amino acid and nucleic acid sequences are considered part of this application and are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0003] The invention relates to compositions comprising, and methods utilizing PBKl protein and DNA.

BACKGROUND OF THE INVENTION

[0004] It was estimated that approximately 150 million people worldwide had type 2 diabetes (T2D) in the year 2000, with the prediction that this number could double to 300 million by 2025 (1). T2D is characterized by insulin resistance in peripheral tissues and deficient β-cell insulin-secretory response to glucose. Glucose-sensing by pancreatic β-cell plays an important role in regulating glucose homeostasis and onset of T2D. Normal pancreatic β-cells are able to sense minor changes in blood glucose levels, and promptly respond to such changes by adjusting insulin secretion rates to maintain normoglycemia (2). In patients with insulin resistance, the pancreatic β-cells have to secret higher levels of insulin as a compensatory response to insulin resistance in order to maintain normoglycemia, resulting in hyperinsulinemia. Consequently, T2D develops only in subjects that are unable to sustain this β-cell compensatory response (3, 4). This is supported by results from longitudinal studies of subjects that develop T2D. These patients show a rise in insulin levels in the normoglycemic and prediabetes phases, followed by a decline in insulin secretion when β cells loss their ability to sense glucose, resulting pancreatic β cell failure and onset of diabetes (5). A longitudinal study in Pima Indians also confirmed that β-cell dysfunction was the major determinant of

progression from normoglycemia to diabetes (6). Furthermore, the natural history of T2D entails progressive deterioration in β-cell function (7) and loss of β cell mass due to apoptosis (8, 9). [0005] Current treatment options for type 2 diabetes include insulin, sulfonylureas, glitinides, acarbose, metformin, thiazolidinediones. These drugs lower blood glucose through diverse mechanisms of action. However, many of the drugs cannot prevent β-cell death or reestablish β-cell mass, and most of the oral hypoglycemic agents lose their efficacy over time, resulting in progressive deterioration in β-cell function and loss of glycemic control. Moreover, sulfonylurea therapy has been shown to induce apoptosis in rodent β-cells (10) or cultured human islets (11), thus likely exacerbating β-cell loss in T2D patients. Consequently, there has been intense interest in the development of therapeutic agents that preserve or restore functional β-cell mass (12). Several agents with the potential to inhibit β-cell apoptosis and/or increase β- cell mass have been identified in preclinical studies (12). One of the agents, a GLP-I analogue, commercially known as Byetta (exenatide), has been shown to lower blood glucose level by improving β-cell function (13-15). Byetta is a peptide derived from the venom of the GiIa monster, a poisonous lizard. Treatment of β-cell with Byetta has been shown to improve β-cell glucose sensing concurrent with preservation of β-cell mass and stimulation of β-cell regeneration (16). However, Byetta must be administered by injection twice daily, and long term usage of the drug has been associated with development of anti-exenatide antibodies in diabetic subjects. Additionally, the drug slows gastric emptying, and causes gastrointestinal discomfort.

[0006] Thus, there is a continuing need for compositions and methods for detection and treatment of diabetes in a subject. Further, development of an oral antidiabetic drug that can improve glucose sensing by pancreatic β-cells is required for treatment of type 2 diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 depicts a northern blot that indicates that PBKl mRNA is most abundantly expressed in human pancreas.

[0008] Figure 2 depicts a western blot that indicates that PBKl is predominantly expressed in endocrine islets, and that PBKl protein expression was significantly up-regulated in islets of diabetic db mice.

[0009] Figure 3 graphically represents insulin levels at indicated glucose concentrations for Min-6 cells comprising vector, PBKl, a dominant negative mutant of PBKl (DN), and a PKA

phosphorylation defective mutant of PBKl (T260A); demonstrating that PBKl overexpression improved glucose-sensing by Min-6 cells.

[0010] Figure 4 depicts RT-PCR and insulin secretion data related to inhibition of PBKl and PBK2 mRNA expression. [0011] Figure 5 graphically represents a time course for oral glucose tolerance in PBKl heterozygous knockout mice and wild type controls.

[0012] Figure 6 depicts western blot data related to subcellular localization of recombinant PBKl.

SUMMARY OF THE INVENTION

[0013] The invention relates to compositions comprising-, and methods utilizing PBKl protein and DNA.

[0014] In one aspect, the invention provides a method of detecting type 1 diabetes in a patient, comprising: reacting a patient sample with a PBKl protein or a fragment thereof under conditions that promote antibody- antigen binding; and detecting the presence of an autoantibody in said sample.

[0015] In one embodiment of the present invention, the PBKl protein is immobilized on a solid support. In another embodiment, the patient sample is selected from the group consisting of: blood, serum, plasma, and a biopsy tissue. In another, the detecting of the autoantibody comprises reacting the autoantibody with an antibody having a detectable label.

[0016] In another aspect, the invention provides a mammalian pancreas-derived cell comprising a recombinant nucleic acid encoding a PBKl protein.

[0017] In one embodiment of the present invention, the cell is a cell of a mammalian pancreas-derived cell line. In another embodiment, the cell is a Min6 cell transfected with a recombinant nucleic acid encoding a PBKl protein.

[0018] In another aspect, the invention provides a method of detecting a PBKl modulator, comprising: contacting PKBl with a compound; and detecting a change in PBKl activity, the change in PBKl activity indicative that the compound is a PBKl modulator. [0019] In a related aspect, the invention encompasses a pharmaceutical composition for treatment of type 2 diabetes in a subject, the composition comprising a modulator of PBKl.

[0020] In a further related aspect, the invention provides a method of identifying a PBKl modulator, comprising one or more of: a) contacting a PBKl protein with a test agent under conditions that promote kinase activity of said PBKl protein, and detecting an increase in the

kinase activity; and b) contacting a cell that expresses a PBKl protein with a test agent under conditions that promote glucose-stimulated insulin secretion from said cell, and detecting an increase in the glucose-stimulated insulin secretion from the cell; wherein said increase in a) or b) each indicates that the test agent is a PBKl modulator. [0021] In one embodiment of the present invention, the method further comprises determining that the increase in the glucose-stimulated insulin secretion comprises potentiation of secretagogue- stimulated insulin secretion from a cell that expresses a PBKl protein. In another embodiment, the cell is a mammalian pancreas-derived cell. In another, the cell is a MIN6 cell. In another, the cell contains a recombinant nucleic acid encoding a PBKl protein. [0022] In another aspect, the invention provides a method of screening for an agent that treats a metabolic disease, said method comprising one or more of:

[0023] a) contacting a PBKl protein with a test agent under conditions that promote binding of said test agent to said PBKl protein, and detecting binding of said test agent to said PBK protein; [0024] b) contacting a PBKl protein with a test agent under conditions that promote kinase activity of said PBKl protein, and detecting an increase in the kinase activity; and [0025] c) contacting a cell that expresses a PBK protein with a test agent under conditions that promote glucose-stimulated insulin secretion from said cell, and detecting an increase in the glucose-stimulated insulin secretion from the cell; wherein said disorder is selected from diabetes, type 2 diabetes, obesity, and diabetic retinopathy.

[0026] In one embodiment of the present invention, the screening method further comprises designating said test agent as an agent that treats said disease. In another embodiment, the screening method further comprises designating said test agent as an agent that treats type 2 diabetes. [0027] In another embodiment, the PBKl protein is selected from a protein having a polypeptide sequence that has at least 73%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the protein having a polypeptide sequence set forth in SEQ ID NO: 2. [0028] In another embodiment, the PBKl protein is selected from a protein having a polypeptide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6.

[0029] In another embodiment, step a) is performed prior to step c). In another, step b) is performed prior to step c). In another, step a) is performed prior to b), and step b) is performed prior to step c).

[0030] In another embodiment, the screening method further comprises determining that the increase in the glucose-stimulated insulin secretion comprises potentiation of secretagogue- stimulated insulin secretion from a cell that expresses a PBKl protein. In another, the cell is a mammalian pancreas-derived cell. In another, the cell is a MIN6 cell. In another, the cell contains a recombinant nucleic acid encoding a PBKl protein.

[0031] In another aspect, the invention comprehends delivery of PBK-I DNA to a subject to stimulate pancreatic beta cell differentiation and/or regeneration.

[0032] In a related aspect, the invention provides a method for stimulating cell differentiation and/or regeneration in a pancreatic beta cell comprising delivering PBK-I DNA to said cell. In one embodiment of the present invention, the DNA is operably linked to a promoter that drives expression of said DNA in said cell. In one embodiment, the promoter is an adenoviral promoter, retroviral promoter, or any promoter that can direct expression of PBKl expression in mammalian cells, tissues, or living body. [0033] In another aspect, the invention comprehends the usage of transgenic mice with targeted deletion or overexpression of the PBK-I gene to test efficacy and specificity of PBK-I modulator compounds.

[0034] In a related aspect, the invention provides a method for in vivo screening for an agent that treats diabetes, said method comprising: a) subjecting a mammal comprising a PBKl knockout genome to a diet that promotes diabetes; b) treating said mammal with a test agent; c) determining blood glucose level of said mammal; and d) determining whether said mammal is glucose tolerant. In one embodiment of the present invention, the animal is heterozygous for the PBKl knockout. In another, the animal is homozygous for the PBKl knockout.

DETAILED DESCRIPTION OF THE INVENTION [0035] PBKl (pancreas brain kinase), is shown herein to play a key role in regulating glucose-sensing. Overexpression of PBKl in MIN6 cells greatly improves their glucose- sensing by more than three fold, much greater than that by GLP-I. Moreover, data detailed herein show that PBKl also enhances insulin secretion stimulated by GLP-I and other insulin secretagogues by improving the stimulus-secretion coupling function of β-cells. It is further demonstrated that PBKl is a "master" regulator of the networks that regulate glucose-sensing, since PBKl is associated with both synaptic-like microvesicles and insulin secretion granules. PBKl is predominantly expressed in brain and pancreas, thus eliminating or reducing potential problems of functionally related side effects of an oral antidiabetic compound.

Various embodiments described herein refer to type 1 diabetes and/or type 2 diabetes. For the purposes of the invention described herein these references to the types of diabetes are made consistently with the 1997 recommendations of the American Diabetes Association expert committee for universal adoption of simplified terminology, with which recommendations the National Institute of Diabetic and Digestive and Kidney Diseases (NIDDK) agreed. Accordingly, for the purposes of the invention described herein, type 1 diabetes encompasses art-recognized references to type I diabetes, juvenile diabetes, insulin-dependent diabetes mellitus, and/or IDDM; and type 2 diabetes encompasses art-recognized references to type II diabetes, adult-onset diabetes, noninsulin-dependent diabetes mellitus, and/or NIDDM. [0036] In accordance with the present invention, various techniques and terms, including, but not limited to, conventional molecular biology, microbiology, immunology and recombinant DNA techniques and terms may be used which are known by those of skill in the art. Such techniques and terms are described and/or defined in detail in standard references such as Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel, F. et al., Current Protocols in Molecular Biology, John Wiley & Sons, 1994; Wild, D., The Immunoassay Handbook, 3rd Ed., Elsevier Science, 2005; Gosling, J. P., Imunoassays: A Practical Approach, Practical Approach Series, Oxford University Press, 2005; and Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; and other references described herein. [0037] PBKl protein provided in compositions and methods described herein may be isolated from natural sources, such as brain or pancreas of an organism. Alternatively, PBKl protein may be generated recombinantly, such as be expression using an expression construct, in vitro or in vivo. Nucleic acid sequences encoding PBKl have been isolated as exemplified by nucleic acid sequences described herein along with amino acid sequences of PBKl. Methods and compositions are not limited to PBKl having the amino acid sequence described herein in detail. Homologs, including orthologs, of PBKl may be used. In addition, as will be appreciated by one of skill in the art, due to the degeneracy of the genetic code, more than one nucleic acid will encode an identical protein. Thus, nucleic acids encoding PBKl or a homolog thereof are not limited to those nucleic acids described herein in detail. [0038] "PBKl protein" means a protein having at least 73%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the protein having a polypeptide sequence set forth in SEQ ID NO: 1, or a protein encoded by a nucleic acid sequence that hybridizes under high stringency hybridization conditions to the nucleic acid set

forth in SEQ ID NO: 1 or a complement thereof so long as the protein effects the function described in the particular inventive method comprising use of the protein. For example, in a method of screening of the invention, a protein encoded by a nucleic acid sequence that hybridizes under high stringency hybridization conditions to the nucleic acid set forth in SEQ ID NO: 1 or a complement thereof is operable in the inventive method so long as the protein effects the described binding, kinase activity, or glucose- stimulated insulin secretion. [0039] High stringency hybridization conditions are known to the ordinarily skilled artisan. For instance, high stringency conditions can be achieved by incubating the blot overnight (e.g., at least 12 hours) with a long polynucleotide probe in a hybridization solution containing 7% SDS, 0.5 M NaPO ₄ , pH 7, 1 mM EDTA at 65 ⁰ C, followed by one or more washes with a 0.1% SDS, Ix SSC solution at 65 ⁰ C. Whereas high stringency washes can allow for less than 5% mismatch, reduced or low stringency conditions can permit up to 20% nucleotide mismatch.. A fragment of PBKl protein is any fragment of a PBKl protein that is operable in the described method utilizing the fragment, as understood by the ordinarily skilled artisan. A fragment of PBKl protein is operative in any of the inventive methods described herein utilizing a PBKl protein. For example, in a method of the invention comprising reacting a patient sample with a PBKl protein or a fragment thereof under conditions that promote antibody- antigen binding, a fragment of the PBKl will be such understood to be operative, so long as the fragment effects the described antibody- antigen binding. Furthermore, it is understood by the ordinarily skilled artisan that such binding refers to specific binding as determinable by use of appropriate controls to distinguish it from nonspecific binding. Similarly, it is understood by the ordinarily skilled artisan that binding of a test agent or modulator to PBKl protein refers to specific binding as determinable by use of appropriate controls to distinguish it from nonspecific binding. [0040] "PBKl DNA" means an isolated DNA molecule having a sequence that has at least 73%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the DNA sequence set forth in SEQ ID NO: 1, or an isolated DNA molecule having a sequence that hybridizes under high stringency hybridization conditions to the nucleic acid set forth in SEQ ID NO: 1 or a complement thereof, so long as the DNA effects the function described in the particular inventive method comprising use of the DNA. PBKl DNA includes an isolated DNA molecule that has a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 18. A fragment of PBKl DNA is any fragment of a PBKl protein that is operable in the described method utilizing the fragment, as understood by the ordinarily skilled artisan. A

fragment of PBKl DNA is operative in any of the inventive methods described herein utilizing a PBKl DNA.

[0041] A "test agent" or "modulator" as described in any of the inventive methods provided herein comprehends a compound; small molecule; biochemical; cytokine; biological including protein, peptide, antibody, or fragments thereof.

[0042] Conditions that promote binding, kinase activity, or glucose- stimulated insulin secretion as described in any of the inventive methods provided herein are well known in the art; including such conditions described in the references provided herein, and otherwise described or illustrated herein. [0043] Methods of detecting type 1 diabetes are provided according to the present invention which include detecting an autoantibody in a patient sample by reacting the patient sample with a PBKl protein. Detection of an autoantibody in the sample is indicative of type 1 diabetes in the patient. In particular embodiments, methods of the present invention for detecting type 1 diabetes are advantageously used to screen patients suspected of developing type 1 diabetes or likely to be susceptible to developing type 1 diabetes. For example, an inventive assay is used in a subject having a medical history or genetic background which predisposes the subject to development of type 1 diabetes.

[0044] Any of various techniques may be used to detect an autoantibody to PBKl in a sample obtained from a subject. Standard immunomethods including, but not limited to, ELISA, radioimmunoassay, immunoblotting, immunoprecipitation assay, or immunodiffusion assay, may be used to detect an autoantibody to PBKl in a sample. Immunomethods are described in detail in standard references such as Wild, D., The Immunoassay Handbook, 3rd Ed., Elsevier Science, 2005; Gosling, J. P., Imunoassays: A Practical Approach, Practical Approach Series, Oxford University Press, 2005; and Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988.

[0045] PBKl protein or its peptide analogues may be immobilized on a solid support for incubation with the patient sample in particular embodiments.

[0046] PBKl protein used in methods of the present invention for detection of type 1 diabetes is full-length PBKl in particular embodiments. In further embodiments, an antigenic fragment of PBKl is used. An antigenic fragment of PBKl is a portion of a PBKl protein which stimulates an immune response in a subject.

[0047] A patient sample is typically a blood, serum, plasma, and/or biopsy tissue sample. A particular biopsy tissue used in a method of the present invention is pancreas biopsy tissue.

In addition to detection of type 1 diabetes, methods and compositions for use in treatment of diabetes are provided according to embodiments of the present invention.

[0048] In a particular embodiment, a mammalian pancreas-derived cell including a recombinant nucleic acid encoding PBKl is provided. A mammalian pancreas-derived cell including a recombinant nucleic acid encoding PBKl may be a primary pancreas cell, such as a pancreatic beta cell or a cell of a mammalian pancreas-derived cell line. In particular embodiments a cell of a mammalian pancreas-derived cell line is a Min6 cell. Further embodiments include any human pancreatic beta cell line including a recombinant nucleic acid encoding PBKl. [0049] A method of treating type 1 diabetes in a subject is provided which includes delivering a mammalian pancreas-derived cell including a recombinant nucleic acid encoding PBKl to a subject. In preferred embodiments, the cell is a glucose-sensing cell competent to secrete insulin. In particular embodiments, the mammalian pancreas-derived cell including a recombinant nucleic acid encoding PBKl is delivered to the pancreas. In further embodiments, the microencapsulated mammalian pancreas-derived cell including a recombinant nucleic acid encoding PBKl is delivered to liver or the kidney capsule of the subject.

[0050] An in vivo animal model for transplantation of exogenous cells expressing a transgene to the pancreas is exemplified by a model of type 1 diabetes generated by treatment of an animal with streptozotocin (STZ). Streptozotocin is well-known as a naturally occurring chemical that is particularly toxic to the insulin-producing beta cells of the pancreas. The STZ- diabetic animals will be transplanted with pancreatic beta cell line stably overexpressing PBKl gene to test efficacy of the transplantation in the treatment of type 1 diabetes. Animals which may be used in a streptozotocin model of type 1 diabetes include, but are not limited to, rodents, rabbits and dogs. [0051] In further embodiments, a nucleic acid encoding PBKl is delivered to a subject to stimulate pancreatic beta cell differentiation and/or regeneration. Pancreatic β-cell differentiation/regeneration is measured by cell number counting and by immunofluorescence analysis of BrdU incorporation in pancreatic β-cell lines overexpressing the PBK-I kinase. [0052] Methods of identifying a PBKl modulator are provided according to embodiments of the present invention which include contacting PKBl with a putative modulator compound and assaying for a change in PBKl activity. For example, a detected increase in PBKl activity is indicative that the putative activator compound is an PBKl activator. A detected decrease in PBKl activity is indicative that the putative inhibitor compound is a PBKl inhibitor. It is

understood by the ordinarily skilled artisan that the detected increase or decrease in any of the inventive methods described herein comprehends reference to appropriate controls for the purposes of the particular method.

[0053] An assay used in methods of identifying a PBKl modulator or in methods of screening described herein may have any of various formats, including, but not limited to, cell- based and array assays. An array assay refers to an ordered array of one or more materials, such as an arrangement of addressable regions including putative activators, for example. [0054] In particular embodiments, the inventive method includes detection of a change in secretagogue- stimulated insulin secretion, such as glucose-stimulated, GLP-I -stimulated and/or KCl- stimulated insulin secretion from a cell expressing PBKl. A cell expressing PBKl is a mammalian pancreas-derived cell in particular embodiments of an inventive method. For example, a cell expressing PBKl for use in an assay for detecting modulated PBKl activity following contact with a putative modulator is a MIN6 cell containing a recombinant nucleic acid encoding PBKl. [0055] Assays for modulator activity or for the screening described herein are optionally performed using a transgenic non-human animal, such as a transgenic mouse, having targeted deletion or overexpression of the PBK-I gene, for example to test efficacy and specificity of PBK-I modulator compounds. Methods for targeted deletion or overexpression of a gene in a cell and/or in an animal, such as a transgenic mouse, are known in the art as exemplified by description in references including, but not limited to, U.S. Patent Nos. 5,994,618 and 6,891,082, Nagy, A. et al., Manipulating the Mouse Embryo: A Laboratory Manual, 3 ^rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2002 and Pinkert, C. A., Transgenic Animal Technology, A Laboratory Handbook, 2 ^nd ed., Academic Press, 2002. See also Kishi M. et al. (2005) Science 37: 929-931. [0056] A pharmaceutical composition for treatment of type 2 diabetes in a subject is provided which includes a modulator of PBKl. A PBKl modulator is an activator or inhibitor of PBK-I activity in particular embodiments.

[0057] In particular embodiments, a PBKl modulator or agent described herein is formulated for oral delivery to a subject, in particular a subject having need of treatment for type 2 diabetes. In further embodiments, the pharmaceutical composition is formulated for parenteral, nasal, topical, ocular, buccal, pulmonary, or rectal delivery to the subject. [0058] Formulation of pharmaceutical compositions for particular routes of administration is known in the art and is described in detail in references such as Allen, L. V. et al., Ansel's

Pharmaceutical Dosage Forms and Drug Delivery Systems, 8 ^th ed., Lippincott, Williams & Wilkins, 2005; and Remington: The Science and Practice of Pharmacy, 21 ^st ed., Lippincott, Williams & Wilkins, 2006.

[0059] The terms "subject" and "patient" are used interchangeably herein and refer to individual animals, particularly mammals, including, but not limited to, humans.

[0060] The invention comprehends PBKl as a drug target to screen compounds to be used for the treatment of type 2 diabetes. In particular embodiments, activators of AMPK (5'-AMP- activated protein kinase), related compounds and derivatives are PBKl activators for the treatment of type 2 diabetes which improve glucose- sensing of the pancreatic beta-cells. [0061] In further embodiments, inhibitors of PBK-I are identified. PBK-I inhibitors are useful, for instance, to prevent/treat hyperglycemia-induced apoptosis of pancreatic beta cells, or used as reference compounds in the methods described herein.

[0062] Embodiments of inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.

EXAMPLES

EXAMPLE 1: Cloning and expression Molecular cloning of PBKl [0063] The coding region of the human PBKl cDNA (SEQ ID NO: 18; NCBI Ace. AF533876) is amplified by PCR from human fetal brain marathon-ready cDNA(BD Bioscience) using primers by 5 ' -CGATGACATCGACGGGGAAGGAC-3 ' (SEQ ID NO: 7) and 5'- GATGGCCTCGTGGAGGTGAC ATG-3' (SEQ ID NO: 8) and subcloned into Srf-1 site of pCR-Script-Amp SK(+) vector. A flag-tagged version of PBKl is generated by PCR amplification by using primer pairs 5'-

GCCACCATGGATTACAAGGATGACGACGATAAGACATCGACGGGGAAGGACGGCG GC-3'(SEQ ID NO: 9), and 5'-GATGGCCTCGTGGAGGTGACATG-S' (SEQ ID NO: 10). The resulting PCR product is cloned into Srf-1 sites of pCR-Script-Amp SK(+) vector, sequenced, and subcloned to the Hindlll-Notl sites of pcDNA3.1. Retrovirus expression vector

[0064] The insert is excised with BamHl-Xhol from pcDNA3.1-PBKl and ligated into the BamHl and Sail sites of pBabe-puro to generate pBabe-PBKl for retroviral expression in MIN6 cells.

Mutants

[0065] Site-directed mutagenesis is performed by using Quickchange multi-site kit (Strategene). Amino acid residue Arg-48(K48M) or Thr-260(T260A) of PBKl was replaced by Met and Ala, respectively. The primers for these two mutants are 5'- CCAGAAGGTGGCCATCATGATCGTCAACCGTGAG-S' (SEQ ID NO: 11) (K48M) and 5'- CGCCGCACGCCGCCTCGCGCTAGAGCACATTCAG-3' (SEQ ID NO: 12) (T260A). The mutants are confirmed by sequencing analysis. Cell culture and retroviral infection [0066] Pancreatic beta-cell line, Min6 cells, described in Miyazaki et al., Endocrinology, 1990 Jul;127(l):126-32, are cultured in DMEM with 15% FBS, 25mM glucose, 100 micromolar beta-mercaptoethanol, 100 units/ml penicillin/streptomycin. For retroviral infection, pBabe-puro constructs are used to transfect 293T-derived phoenix cells by the calcium phosphate method. Two days after transfection, supernatants containing viral particles are harvested and used to infect MIN6 cells. The cells are then selected by lmicrogram/ml puromycin. EXAMPLE 2: Human tissue distribution of PBKl mRNA.

[0067] Tissue distribution of PBKl (pancreas brain kinase-1), also known as BRSK2, SAD-A, SADl, STK29, PENIlB, Cllorf7 (18, 19) is examined. Northern blot analysis shows that PBKl is almost exclusively expressed in human pancreas and brain, and most abundantly in human pancreas (Figure 1); implicating an important role for PBKl in regulating pancreas function. Similar results were obtained when heart tissue was included in Northern blots. Northern blot analysis was carried out using multi-tissue blot from Clontech and radio-labeled human PBKl as a probe.

EXAMPLE 3: PBKl protein is predominantly expressed in endocrine islets. [0068] PBKl protein expression were analyzed by western blot analysis using mouse monoclonal antibody to PBKl and protein lysate from Min6 cells, pancreatic islets and exocrine pancreas isolated from C57B16 (B16), non-diabetic control db mouse (Db-C), hyperglycemic db mouse (Db-HG), or db mouse under normalglycemia (Db-LG), respectively. The same blot was also used to analyze the expression level of β-actin as an internal control for sample loading. The results showed that PBKl is predominantly expressed in endocrine islet, but not detectable in exocrine pancreas. The results also showed that PBKl protein expression was significantly up-regulated in islets of diabetic db mice. See Figure 2. EXAMPLE 4: PBKl overexpression improved glucose-sensing by Min-6 cells.

[0069] Min-6 is a stable cell line derived from mouse pancreatic beta cells (25). In comparison with other pancreatic beta-cell lines, MIN6 cells maintain the integrity of glucose- sensing ability. To achieve high transfection rates for the expression of PBKl in MIN6 cells, a recombinant retroviral expression system was created for PBKl as described above, and a flag tagged expression vector for PBKl is generated to facilitate analysis of its expression in MIN6 cells, a mouse islet beta-cell line. As shown in Figure 3, overexpression of PBKl in MIN6 cells significantly enhances glucose and KCl stimulated insulin secretion. The effect of PBKl on insulin secretion is fully dependent on its kinase activity. [0070] The Min-6 cells were cultured in DMEM medium containing 4.5 g/1 glucose and L- glutamine, and were infected with recombinant retroviruses overexpressing the human PBKl, a dominant negative mutant of PBKl (DN), or a PKA phosphorylation defective mutant of PBKl (T260A). The dominant negative mutant was generated by replacing lysine at aa 48 with methionine. The PKA defective mutant was generated by replacing threonine at aa 260 with an alanine. The infected Min-6 cells were selected for puromycin resistance as a stable pool. For measurement of glucose sensing by Min-6 cells, the stable pool of the infected Min-6 cells were preincubated for 1 hour at 37 oC in Krebs Ringer buffer containing 0 mM glucose, followed by 2 hour incubation with the indicated concentration of 2.8 mM, 16.7 mM, and 28 mM of glucose or 50 mM KCl. The supernatants were then collected and analyzed for insulin levels by radioimmuno assays using a kit from Linco Research, Inc. With respect to DN, mutation of the key amino acids for the kinase activation of PBK completely abolished its effect on insulin secretion. With respect to T260A, the mutant PBKl partially lost its ability to improve insulin secretion by glucose and KCl (Figure 3). The results showed that the wild type PBKl, but not the mutants, potentiated glucose- and KCl-stimulated insulin secretion. EXAMPLE 5: PBKl deficiency impaired glucose-stimulated insulin secretion from Min-6 cells.

[0071] Endogenous mouse PBKl ( also known as SAD-A; BRSK2) and PBK2 (also known as SAD-B; BRSKl) mRNA expression was inhibited by transient transfection of Min-6 cells with 200 nM of siRNAs targeting the coding region of the mouse PBKl (194-214: 5'- AGCGAGAGATTGCCATCTTGA-3') (SEQ ID NO: 16) and PBK2 (468-488: 5- GCCAGAGAACCTGCTGTTGGA-3') (SEQ ID NO: 17) coding regions or a scrambled- sequence siRNA as a negative control (Dharmacon) using Lipofectamine 2000 (Invitrogen). Efficiency of PBKl and PBK2 knockdown were assessed by RT-PCR. The results showed more than 70% reduction of the endogenous PBKl and PBK2 mRNA expression (A). Sixty hours

after the transfection, insulin secretion was analyzed from the Min-6 cells as described in Figure 4. The results showed that deficiency of PBKl, but not PBK2, resulted in impaired glucose sensing by Min-6 cells (B).

EXAMPLE 6: PBKl deficiency in mice caused diabetes. [0072] Oral glucose tolerance test were carried out in awakening PBKl heterozygous knockout mice and the wild type controls to analyze the onset of diabetes. The heterozygous PBKl knockout (n = 4) and the wild type control mice (n = 4) at three months of age on regular chow were fasted overnight and then analyzed for blood glucose levels using blood samples collected by tail bleeding (0 min). The mice were then given 50% glucose solution by oral gavage at 2.5 g glucose/kg body weight. Blood samples were collected from tail-bleeding at 30, 60, and 120 minutes, respectively, after the glucose overload and analyzed for glucose levels by glucose meter. The results (Figure 5) showed that the heterozygous PBKl knockout mice have higher fasting blood glucose level (panel A). One of the heterozygous mice developed diabetes as judged by the fasting blood glucose level and glucose tolerance test (panel B). EXAMPLE 7: Subcellular fractionation of PBKl

[0073] PBKl is associated with both synaptic-like microvesicles and insulin secretion granules. Subcellular localization of the recombinant PBKl in MIN-6 cells was analyzed by sucrose gradient and Western blot analysis using markers for synaptic-like microvesicles and insulin secretion granules. As shown by Figure 6, the recombinant PBKl is co-localized with both Rim2 and VAMP2 in subcellular fractions segregated by sucrose gradient. Rim2 is a putative effector protein for Rab3s, synaptic GTP -binding proteins. RIM2 is localized close to the active zone at the synapse and regulates neurotransmitter releases (20). In insulin- secreting β-cells, Rim2 interacts with cAMP-binding protein cAMP-GEFII (or Epac 2), and mediates cAMP-dependent, PKA-independent insulin secretion (21). VAMP2 is associated with insulin secretory granules and regulates insulin exocytosis from pancreatic β-cells (22). The data support a dynamic role of PBKl in regulating insulin secretion by interacting with proteins of different networks involved in insulin secretion.

[0074] Sucrose gradient fractionation is performed by the method of Mizuta et all (17). Briefly, a stable MIN6 cell clone overexpressing PBKl is harvested by homogenization buffer containing 200 mM sucrose, 50 mM NaCl, 2mM EGTA, 1OmM HEPES at pH7.2, and ImM phenylmethylsulfony fluoride and homogenized. The homogenate is centrifuged at 1770 g for 6 min at 4 oC. The resulting postnuclear supernatant is applied to the top of sucrose gradients (0.4, 0.6, 0.8, 1.0, 1.4, 1.8 M) in 1OmM HEPES, pH 7.2, and 2mM EGTA, and centrifuged at

55,00Og for 2 hours at 4 oC. The fractions are collected from the top to bottom, precipitated with 15% trichloroacetic acid, and subjected to immunoblot analysis using anti-Flag antibody(Sigma), anti-Rim2 antibody (Synaptic Systems), and anti-VAMP2 antibody (Calbiochem). EXAMPLE 8: Polymorphism analysis for early diagnosis of type 1 diabetes and type 2 diabetes

[0075] PBKl is localized on chromosome Ilpl5.5, an IDDM2 diabetic locus. Sequence analysis of the 3 '-untranslated region revealed a polymorphism, described in Miura et al. (18), which can be used to predict the onset of type 2 diabetes. The polymorphism will be investigated by analyzing the patterns of DNA fragments from Hhal/BstUI digestion of genomic DNAs of the 3' untranslated region of PBKl gene from diabetic and non-diabetic subjects. The genomic DNAs from diabetic and non-diabetic subjects will be amplified by PCR reactions (35 cycles of 94 ⁰ C for 30 seconds, 59 ⁰ C for 30 seconds, and 72 ⁰ C for 90 seconds) using a primer pair: 5'-GTCACCTGACCCCTCAGCAA-S' (SEQ ID NO: 13) and 5'- CACCAGCTCTGTCCTCAGAG-S' (SEQ ID NO: 14). The PCR product will be purified by Qiaquick PCR purification kit (Qiagen), digested with Hhal/BstUI, and separated on a 6% polyacrylamide gel.

EXAMPLE 9: Early diagnosis of Type 1 diabetes: [0076] Type 1 diabetes is caused by autoimmune destruction of pancreatic beta cells, which generally occurs over a period of several years leading to the eventual onset of overt diabetes mellitus. During this extended pre-clinical phase, the characteristic circulation of autoantibodies against a variety of islet cell antigens including glutamic acid decarboxylase (GAD65), IA-2 (a tyrosine phosphatase-like protein), and insulin provides early markers of autoimmune disease activity as well as early diagnosis of type 1 diabetes. The present invention comprehends PBKl as an autoantigen in type 1 diabetes, and that the autoantigen can be used as a marker for early diagnosis of type 1 diabetes by using enzyme-linked immunosorbent assay (ELISA), radioimmuno assays (RIA), and the like. In the ELISA assay, purified PBKl protein will be used to detect the presence of antoantibodies to PBKl from serum of type 1 diabetic patients. Methods for detecting presence or absence of autoantigens are known to the ordinarily skilled artisan. For example, such methods have been applied to detect presence or absence of GAD65 and other autoantigens (23). EXAMPLE 10: Application to Type 2 diabetes.

[0077] PBKl can be used as a drug target to screen activator compounds for the treatment of diabetes, obesity, diabetic retinopathy, and other metabolic diseases. The screening process

illustratively comprehends binding assay, kinase assay, and functional assay. PBKl activators can be identified by measuring the binding potency of the compounds to the kinase ATP binding site through competition with a conjugated binding probe as described in reference 24. PBKl activators are expected to stimulate kinase activity in a kinase assay. In the PBKl kinase assay, purified PBKl from E. coli or sf-9 insect cells are incubated in a kinase buffer (Cell Signaling Technology, USA) in the presence of 0.5 mM LNR peptide (KKLNRTLSFAEPG) (SEQ ID NO: 15) in the presence of 10 μM ATP, and 1 μl [γ- ³² P]-ATP (5 μCi). After incubation at 30 ⁰ C for 30 min, 15 μl aceticacidglacial is added to stop the reaction. The kinase activity is quantified by radioactivity incorporated into the LNR peptide using phosphocellulose P-81 filter squares. For high throughput kinase assay for PBKl activators, purified PBKl protein is incubated with sepharose beads coupled with LNR peptide in the kinase buffer in the presence of 0.2mM of [γ- ³² P]-ATP for 30 min, and then washed three times before scintillation counting of radioactivity. In the functional assay, PBKl activators are expected to potentiate glucose stimulated insulin secretion from isolated islet beta cells or cultured pancreatic beta cell lines, such as Min-6 cells. In this assay, addition of a PBKl activator potentiates glucose stimulated insulin secretion in a glucose-dependent manner, but is ineffective in the absence of glucose, as demonstrated by Figure 3. EXAMPLE 11: Sequence data [0078] SEQ ID NO. ' s: 1 and 2 are with reference to: Accession NO. NM_003957; 3506 bp; Homo sapiens BR serine/threonine kinase 2 (BRSK2) setting forth:

/translation="MTSTGKDGGAQHAQYVGPYRLEKTLGKGQTGLVKLGVHCVTCQ K

VAIKIVNREKLSESVLMKVEREIAILKLIEHPHVLKLHDVYENKKYLYLVLEHVSGG E LFDYLVKKGRLTPKEARKFFRQI ISALDFCHSHSICHRDLKPENLLLDEKNNIRIADF GMASLQVGDSLLETSCGSPHYACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDD DNLRQLLEKVKRGVFHMPHFIPPDCQSLLRGMIEVDAARRLTLEHIQKHIWYIGGKNE

PEPEQPIPRKVQIRSLPSLEDIDPDVLDSMHSLGCFRDRNKLLQDLLSEEENQEKMI Y FLLLDRKERYPSQEDEDLPPRNEIDPPRKRVDSPMLNRHGKRRPERKSMEVLSVTDGG SPVPARRAIEMAQHGQRSRSISGASSGLSTSPLSSPRVTPHPSPRGSPLPTPKGTPVH TPKESPAGTPNPTPPSSPSVGGVPWRARLNSIKNSFLGSPRFHRRKLQVPTPEEMSNL TPESSPELAKKSWFGNFISLEKEEQIFWIKDKPLSSIKADIVHAFLSIPSLSHSVIS

QTSFRAEYKATGGPAVFQKPVKFQVDITYTEGGEAQKENGIYSVTFTLLSGPSRRFK R WETIQAQLLSTHDPPAAQHLSDTTNCMEMMTGRLSKCGIIPKS"

ORIGIN

1 gctgttcggc tcagctgcac ggctcggctc ggctcggctc ggctcggctg cgcggccgct

61 gacgggcgtg cgctgggggc gcggggcgcg gggcgcgggc ctcggcggcg gcggcggcgg

121 cggcggcgga agccaggtgc ccccgcccgc cctgtcctct cgacgaggcg gaggcgtcgc 181 cgcgggccag gcctcggact gccgcgtcgg agtggacgcg gggggcggcg gcgcgggcgg

241 acgcgggcgg cgcgaagcag cggggcccgc gggggcgccc cggccgggtc ggcgcggacg

301 gcactcggcg gacgcgggcg gacgctgggc ggcccctccc tgcccgcgcg cccgggcgcc

361 cctggccggc gccgggcccc agagcgatga catcgacggg gaaggacggc ggcgcgcagc

421 acgcgcagta tgttgggccc taccggctgg agaagacgct gggcaagggg cagacaggtc 481 tggtgaagct gggggttcac tgcgtcacct gccagaaggt ggccatcaag atcgtcaacc

541 gtgagaagct cagcgagtcg gtgctgatga aggtggagcg ggagatcgcg atcctgaagc

601 tcattgagca cccccacgtc ctaaagctgc acgacgttta tgaaaacaaa aaatatttgt

661 acctggtgct agaacacgtg tcaggtggtg agctcttcga ctacctggtg aagaagggga

721 ggctgacgcc taaggaggct cggaagttct tccggcagat catctctgcg ctggacttct 781 gccacagcca ctccatatgc cacagggatc tgaaacctga aaacctcctg ctggacgaga

841 agaacaacat ccgcatcgca gactttggca tggcgtccct gcaggttggc gacagcctgt

901 tggagaccag ctgtgggtcc ccccactacg cctgccccga ggtgatccgg ggggagaagt

961 atgacggccg gaaggcggac gtgtggagct gcggcgtcat cctgttcgcc ttgctggtgg

1021 gggctctgcc cttcgacgat gacaacttgc gacagctgct ggagaaggtg aagcggggcg 1081 tgttccacat gccgcacttt atcccgcccg actgccagag tctgctacgg ggcatgatcg

1141 aggtggacgc cgcacgccgc ctcacgctag agcacattca gaaacacata tggtatatag

1201 ggggcaagaa tgagcccgaa ccagagcagc ccattcctcg caaggtgcag atccgctcgc

1261 tgcccagcct ggaggacatc gaccccgacg tgctggacag catgcactca ctgggctgct

1321 tccgagaccg caacaagctg ctgcaggacc tgctgtccga ggaggagaac caggagaaga 1381 tgatttactt cctcctcctg gaccggaaag aaaggtaccc gagccaggag gatgaggacc

1441 tgcccccccg gaacgagata gaccctcccc ggaagcgtgt ggactccccg atgctgaacc

1501 ggcacggcaa gcggcggcca gaacgcaaat ccatggaggt gctcagcgtg acggacggcg

1561 gctccccggt gcctgcgcgg cgggccattg agatggccca gcacggccag aggtctcggt

1621 ccatcagcgg tgcctcctca ggcctttcca ccagcccact cagcagcccc cgggtgaccc 1681 ctcacccctc accaaggggc agtcccctcc ccacccccaa ggggacacct gtccacacgc

1741 caaaggagag cccggctggc acgcccaacc ccacgccccc gtccagcccc agcgtcggag

1801 gggtgccctg gagggcgcgg ctcaactcca tcaagaacag ctttctgggc tcaccccgct

1861 tccaccgccg gaaactgcaa gttccgacgc cggaggagat gtccaacctg acaccagagt

1921 cgtccccaga gctggcgaag aagtcctggt ttgggaactt catcagcctg gagaaggagg

1981 agcagatctt cgtggtcatc aaagacaaac ctctgagctc catcaaggct gacatcgtgc

2041 acgccttcct gtcgattccc agtctcagcc acagcgtcat ctcccaaacg agcttccggg

2101 ccgagtacaa ggccacgggg gggccagccg tgttccagaa gccggtcaag ttccaggttg

2161 atatcaccta cacggagggt ggggaggcgc agaaggagaa cggcatctac tccgtcacct 2221 tcaccctgct ctcaggcccc agccgtcgct tcaagagggt ggtggagacc atccaggccc

2281 agctgctgag cacacacgac ccgcctgcgg cccagcactt gtcagacacc actaactgta

2341 tggaaatgat gacggggcgg ctttccaaat gtggaattat cccgaaaagt taacatgtca

2401 cctccacgag gccatcctct gtgaccgaag gcagctgctg cggacccgcc ctccctccgc

2461 tcctgctgtt gctgccgggc agtgaggccc agcccagcgc cccgtccacc ccgcggcagc 2521 tcctcgcctc agctccgcac ggcccgtggg aggaaggcca ggctcggggg agcctcctcc

2581 agcccggccg acccggactc ccggtcacct gacccctcag caagaacagc ctgcctggtg

2641 gccttctggg gccaggaccc ctggtgggca acgtagccac aggaacaggc cccgtccacc

2701 gcctccacgc cgcacctgga ggcctcctcg caggcccgtg ccccgcctcc ctggctgcgc

2761 cgcctccgtg tagtcttggc ctcctcaggc tgcctcccgt cctctcgtct cacccgcgcc 2821 tcccttgcct catctggggc ggctgtgggc tctggcgctc ctctctggct gaggtggaaa

2881 cagagacacc ctgcggcacc agagccttcc cagcaggcca ggccgctggg ctgggatcag

2941 tgttatttat ttgccgtttt aatttatgga ttctccgcac ctctgttcag ggaagggcgg

3001 cggccacatc ccctgccgtc tgcgtgtctc aggcagtggg ggggctgggg ccagggcgcc

3061 ctctgaggac agagctggtg gggcgcgggg gggctggcga gctactgtaa actttaaaga 3121 attcctgcaa gatattttta taaacttttt tttcttggtg gtttttggaa aagggtgtgg

3181 gggtgggggc gccgctgggg cagggccagg ttttgtgttt tagtcccttg ctcctgcttc

3241 tttctacaca cacatctaaa gacggtgcgg ctcgctctgt catgggttcc gtctctctgt

3301 ggagaagcag ctccacctct gggggggctc ggggcagagg ggcggtgtct cgtagcgggc

3361 ggcagcgcca gcgcccctct gtcaggctgg ggcaatcttg gttttgtgtc caaaggtgaa 3421 ggggtaggag gagggccctc agctggccct ccccacacac aggacggcag gggcactgtg 3481 aggcttttct tattaaaatg aaaaaa

[0079] SEQ ID NO.'s: 3 and 4 are with reference to: Accession NO. NM_032430; 3109 bp; Homo sapiens BR serine/threonine kinase 1 (BRSKl) setting forth: /translat 1On= ¹¹ MSSGAKEGGGGSPAYHLPHPHPHPPQHAQYVGPYRLEKTLGKGQ

TGLVKLGVHCITGQKVAIKIVNREKLSESVLMKVEREIAILKLIEHPHVLKLHDVYE N KKYLYLVLEHVSGGELFDYLVKKGRLTPKEARKFFRQIVSALDFCHSYSICHRDLKPE NLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPHYACPEVIKGEKYDGRRADMWSCG

VILFALLVGALPFDDDNLRQLLEKVKRGVFHMPHFIPPDCQSLLRGMIEVEPEKRLS L EQIQKHPWYLGGKHEPDPCLEPAPGRRVAMRSLPSNGELDPDVLESMASLGCFRDRER LHRELRSEEENQEKMIYYLLLDRKERYPSCEDQDLPPRNDVDPPRKRVDSPMLSRHGK RRPERKSMEVLSITDAGGGGSPVPTRRALEMAQHSQRSRSVSGASTGLSSSPLSSPRS PVFSFSPEPGAGDEARGGGSPTSKTQTLPSRGPRGGGAGEQPPPPSARSTPLPGPPGS PRSSGGTPLHSPLHTPRASPTGTPGTTPPPSPGGGVGGAAWRSRLNSIRNSFLGSPRF HRRKMQVPTAEEMSSLTPESSPELAKRSWFGNFISLDKEEQIFLVLKDKPLSSIKADI VHAFLSIPSLSHSVLSQTSFRAEYKASGGPSVFQKPVRFQVDISSSEGPEPSPRRDGS GGGGIYSVTFTLISGPSRRFKRVVETIQAQLLSTHDQPSVQALADEKNGAQTRPAGAP PRSLQPPPGRPDPELSSSPRRGPPKDKKLLATNGTPLP"

ORIGIN

1 gggggccggc cagaaacggg ctggggaggg ggggccccgc agcccccctg ggccatgctg

61 actcccgggg cctgaccccc ccgggccagc cccccctccc ccagctccgc ggcccgccga

121 ctgggggggg ccagcccagc cccctgggga cccccggaga ggtggggggc agccgggggg

181 gccgggacgg agcggtcgcc ggcccccacc ggagagacgg ggcgacggcc gcaggggggg

241 cggccggggg accggtcggg ccgggaccaa gggcaccatg tcgtccgggg ccaaggaggg

301 aggtgggggc tctcccgcct accacctccc ccacccccac ccccacccac cccagcacgc

361 ccaatatgtg ggcccctatc ggctggagaa gacgctgggc aaaggacaga cagggctggt

421 taaactcggg gtccactgca tcacgggtca gaaggtcgcc atcaagatcg tgaaccggga

481 gaagctgtcg gagtcggtgc tgatgaaggt ggagcgggag atcgccatcc tgaagctcat

541 cgaacaccca catgtcctca agctccacga cgtctacgag aacaagaaat atttgtacct

601 ggttctggag cacgtctcgg ggggtgagct attcgactac ctggtaaaga aggggagact

661 gacgcccaag gaggcccgaa agttcttccg ccagattgtg tctgcgctgg acttctgcca

721 cagctactcc atctgccaca gagacctaaa gcccgagaac ctgcttttgg atgagaaaaa

781 caacatccgc attgcagact tcggcatggc gtccctgcag gtgggggaca gcctcctgga

841 gaccagctgc gggtcccccc attatgcgtg tccagaggtg attaaggggg aaaaatatga

901 tggccgccgg gcagacatgt ggagctgtgg agtcatcctc ttcgccctgc tcgtgggggc

961 tctgcccttt gatgacgaca acctccgcca gctgctggag aaggtgaaac ggggcgtctt

1021 ccacatgccc cacttcattc ctccagattg ccagagcctc ctgaggggaa tgatcgaagt

1081 ggagcccgaa aaaaggctca gtctggagca aattcagaaa catccttggt acctaggcgg

1141 gaaacacgag ccagacccgt gcctggagcc agcccctggc cgccgggtag ccatgcggag

1201 cctgccatcc aacggagagc tggaccccga cgtcctagag agcatggcat cactgggctg

1261 cttcagggac cgcgagaggc tgcatcgcga gctgcgcagt gaggaggaga accaagaaaa

1321 gatgatatat tatctgcttt tggatcggaa ggagcggtat cccagctgtg aggaccagga

1381 cctgcctccc cggaatgatg ttgacccccc ccggaagcgt gtggattctc ccatgctgag

1441 ccgtcacggg aagcggcgac cagagcggaa gtccatggaa gtcctgagca tcaccgatgc

1501 cgggggtggt ggctcccctg tacccacccg acgggccttg gagatggccc agcacagcca 1561 gagatcccgt agcgtcagtg gagcctccac gggtctgtcc tccagccctc taagcagccc

1621 aaggagtccg gtcttttcct tttcaccgga gccgggggct ggagatgagg ctcgaggcgg

1681 gggctccccg acttccaaaa cgcagacgct gccttctcgg ggccccaggg gtgggggcgc

1741 cggggagcag cccccgcccc ccagtgcccg ctccacaccc ctgcccggcc ccccaggctc

1801 cccgcgctcc tctggcggga cccccttgca ctcgcctctg cacacgcccc gggccagtcc 1861 caccgggacc ccggggacaa caccaccccc cagccccggc ggtggcgtcg ggggagccgc

1921 ctggaggagt cgtctcaact ccatccgcaa cagcttcctg ggctcccctc gctttcaccg

1981 gcgcaagatg caggtcccta ccgctgagga gatgtccagc ttgacgccag agtcctcccc

2041 ggagctggca aaacgctcct ggttcgggaa cttcatctcc ttggacaaag aagaacaaat

2101 attcctcgtg ctaaaggaca aacctctcag cagcatcaaa gcagacatcg tccatgcctt 2161 tctgtcgatc cccagcctga gtcacagtgt gctgtcacag accagcttca gggccgagta

2221 caaggccagt ggcggcccct ccgtcttcca aaagcccgtc cgcttccagg tggacatcag

2281 ctcctctgag ggtccagagc cctccccgcg acgggacggc agcggaggtg gtggcatcta

2341 ctccgtcacc ttcactctca tctcgggtcc cagccgtcgg ttcaagcgag tggtggagac

2401 catccaggca cagctcctga gcactcatga ccagccctcc gtgcaggccc tggcagacga 2461 gaagaacggg gcccagaccc ggcctgctgg tgccccaccc cgaagcctgc agcccccacc

2521 cggccgccca gacccagagc tgagcagctc tccccgccga ggccccccca aggacaagaa

2581 gctcctggcc accaacggga cccctctgcc ctgaccccac ggggccgggg agggagggga

2641 cccccctcca ccccccttcc gtgcccccca actgtgaatc tgtaaataag gcccaaggaa

2701 catgtcggga ggggggtgga cacaaaaacc ggccttgccc tgcagggatg gggctccaca 2761 ggccgtgccc aactgggggt ggttctaggg gaacaggggg cgggggagct gtttctattt

2821 tatttattga ttaatttatt attttattta ttgatcaatc tctctgcggg gtggggtggg

2881 ggagggacgg gagctggttg gggtggctta gcagatccgg acagggccct ctgtccctgt

2941 gtcgtcccca accccctctt cccgggcccc tcctcccctg gtcctccccc cacgaccttc

3001 tgtacggatt tgctctccgg aaggaattct ggtttcgcgt gatcctgcct gcgtccgtgt 3061 ctctgattcc gccggcggca aaaaaaaaaa aaaaaaaaaa aaaaaaaaa

[0080] SEQ ID NO' s. 5 and 6 are with reference to:

NEDO human cDNA sequencing project; 3576 b . p . ; Ninomiya, K . et al.

/organism="Homo sapiens"

/mol_tγpe= "mRNA" /db_xref=" taxon : 9606 " /clone=" BRAMY3018357 " /t is sue_tγpe=" amygdala " /clone_l ib=" BRAMY3 " /note= " clonmg vector : pME 18 SFL3 " CD S 254 . . 2 554

/note= "unnamed prote in product " /codon_start=l /prote m_id= "BAD 18 671 . 1 " /db_xref=" GI : 47077575 "

/translat 1On= ¹¹ MSPEGHPSRWARPRRPCICPSSLCSPREPRSGPAVGRGGAAHHR

VPAGHTPGPQLLQPHLHLPQGQTWLCLQPSPAGLVKLGVHCVTCQKVAIKIVNREKL S ESVLMKVEREIAILKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLT

PKEARKFFRQIISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSL L ETSCGSPHYACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKR GVFHMPHFIPPDCQSLLRGMIEVDAARRLTLEHIQKHIWYIGGKNEPEPEQPIPRKVQ IRSLPSLEDIDPDVLDSMHSLGCFRDRNKLLQDLLSEEENQEKMIYFLLLDRKERYPS QEDEDLPPRNEIDPPRKRVDSPMLNRHGKRRPERKSMEVLSVTDGGSPVPARRAIEMA

QHGQRSRSISGASSGLSTSPLSSPRVTPHPSPRGSPLPTPKGTPVHTPKESPAGTPN P TPPSSPSVGGVPWRARLNSIKNSFLGSPRFHRRKLQVPTPEEMSNLTPESSPELAKKS WFGNFISLEKEEQIFWIKDKPLSSIKADIVHAFLSIPSLSHSVISQTSFRAEYKATG GPAVFQKPVKFQVDITYTEGGEAQKENGIYSVTFTLLSGPSRRFKRVVETIQAQLLST HDPPAAQHLSDTTNCMEMMTGRLSKCDEKNGQAAQAPSTPAKRSAHGPLGDSAAAGPG

PGGDAEYPTGKDTAKMGPPTARREQP "

ORIGIN

1 agtctcaggc tggctagttc ctccttcctg gtcactgagc cagccttgct gaggggagag

61 cgggttctgg acgtgctctg agcttccttc ctcacagcct tgctcctggg ccagatcagc

121 aggaaagcag ccagtgcccc gccatggcct gcccgggtgg ggtcctgaag ctggggccgg

181 agcagggggc acagttctgc cccatctggc cctagtttgg ggagggagcc tggtagggca

241 ccagcctcac cccatgagcc ctgagggcca ccccagccga tgggcacgtc cccgccggcc

301 ctgcatctgt ccttcctccc tctgctcccc aagagagccc aggtctggcc cagcggtggg

361 caggggaggg gccgcacatc acagagtgcc agctggccac actcccggcc cacagctgct

421 ccagccgcac ctccaccttc ctcaaggcca gacctggctc tgcctgcagc ccagcccagc

481 aggtctggtg aagctggggg ttcactgcgt cacctgccag aaggtggcca tcaagatcgt

541 caaccgtgag aagctcagcg agtcggtgct gatgaaggtg gagcgggaga tcgcgatcct

601 gaagctcatt gagcaccccc acgtcctaaa gctgcacgac gtttatgaaa acaaaaaata

661 tttgtacctg gtgctagaac acgtgtcagg tggtgagctc ttcgactacc tggtgaagaa

721 ggggaggctg acgcctaagg aggctcggaa gttcttccgg cagatcatct ctgcgctgga

781 cttctgccac agccactcca tatgccacag ggatctgaaa cctgaaaacc tcctgctgga

841 cgagaagaac aacatccgca tcgcagactt tggcatggcg tccctgcagg ttggcgacag

901 cctgttggag accagctgtg ggtcccccca ctacgcctgc cccgaggtga tccgggggga

961 gaagtatgac ggccggaagg cggacgtgtg gagctgcggc gtcatcctgt tcgccttgct

1021 ggtgggggct ctgcccttcg acgatgacaa cttgcgacag ctgctggaga aggtgaagcg

1081 gggcgtgttc cacatgccgc actttatccc gcccgactgc cagagtctgc tacggggcat

1141 gatcgaggtg gacgccgcac gccgcctcac gctagagcac attcagaaac acatatggta

1201 tatagggggc aagaatgagc ccgaaccaga gcagcccatt cctcgcaagg tgcagatccg

1261 ctcgctgccc agcctggagg acatcgaccc cgacgtgctg gacagcatgc actcactggg

1321 ctgcttccga gaccgcaaca agctgctgca ggacctgctg tccgaggagg agaaccagga

1381 gaagatgatt tacttcctcc tcctggaccg gaaagaaagg tacccgagcc aggaggatga

1441 ggacctgccc ccccggaacg agatagaccc tccccggaag cgtgtggact ccccgatgct

1501 gaaccggcac ggcaagcggc ggccagaacg caaatccatg gaggtgctca gcgtgacgga

1561 cggcggctcc ccggtgcctg cgcggcgggc cattgagatg gcccagcacg gccagaggtc

1621 tcggtccatc agcggtgcct cctcaggcct ttccaccagc ccactcagca gcccccgggt

1681 gacccctcac ccctcaccaa ggggcagtcc cctccccacc cccaagggga cacctgtcca

1741 cacgccaaag gagagcccgg ctggcacgcc caaccccacg cccccgtcca gccccagcgt

1801 cggaggggtg ccctggaggg cgcggctcaa ctccatcaag aacagctttc tgggctcacc

1861 ccgcttccac cgccggaaac tgcaagttcc gacgccggag gagatgtcca acctgacacc

1921 agagtcgtcc ccagagctgg cgaagaagtc ctggtttggg aacttcatca gcctggagaa

1981 ggaggagcag atcttcgtgg tcatcaaaga caaacctctg agctccatca aggctgacat

2041 cgtgcacgcc ttcctgtcga ttcccagtct cagccacagc gtcatctccc aaacgagctt

2101 ccgggccgag tacaaggcca cgggggggcc agccgtgttc cagaagccgg tcaagttcca

2161 ggttgatatc acctacacgg agggtgggga ggcgcagaag gagaacggca tctactccgt

2221 caccttcacc ctgctctcag gccccagccg tcgcttcaag agggtggtgg agaccatcca 2281 ggcccagctg ctgagcacac acgacccgcc tgcggcccag cacttgtcag acaccactaa 2341 ctgtatggaa atgatgacgg ggcggctttc caaatgtgac gagaagaacg ggcaggcggc 2401 ccaggccccc agcacgcccg ccaagcggag tgcccacggc ccactcggtg actccgcggc 2461 cgctggccct ggccccggag gggacgccga gtacccaacg ggcaaggaca cggccaagat 2521 gggcccgccc accgcccgcc gcgagcagcc ttagacacac tagccccccc ccccagcaca 2581 gcactgacag cggctgcctc gccgcccgcc gcccgccctg ccccgagtgg acccgcggcc 2641 gcgccgcccg tccgtccaga ctgttctcag agcctgggag gaaaggaaag gggcgttggg 2701 gccggcctgt gggctgcgcc acccgcgccc gctctctttt ctctctgtct ctgcctctgc 2761 ctgtctctga cagcatcgct tgtttccact ctgataccag gaattatccc gaaaagttaa 2821 catgtcacct ccacgaggcc atcctctgtg accgaaggca gctgctgcgg acccgccctc 2881 cctccgctcc tgctgttgct gccgggcagt gaggcccagc ccagcgcccc gtccaccccg 2941 cggcagctcc tcgcctcagc tccgcacggc ccgtgggagg aaggccaggc tcgggggagc 3001 ctcctccagc ccggccgacc cggactcccg gtcacctgac ccctcagcaa gaacagcctg 3061 cctggtggcc ttctggggcc aggacccctg gtgggcaacg tagccacagg aacaggcccc 3121 gtccaccgcc tccacgccgc acctggaggc ctcctcgcag gcccgtgccc cgcctccctg 3181 gccgcgccgc ctccgtgtag tcttggcctc ctcaggctgc ctcccgtcct ctcgtctcac 3241 ccgcgcctcc cttgcctcat ctggggcggc tgtgggctct ggcgctcctc tctggctgag 3301 gtggaaacag agacaccctg cggcaccaga gccttcccag caggccaggc cgctgggctg 3361 ggatcagtgt tatttatttg ccgttttaat ttatggattc tccgcacctc tgttcaggga 3421 agggcggcgg ccacatcccc tgccgtctgc gtgtctcagg cagtgggggg gctggggcca 3481 gggcgccctc tgaggacaga gctggtgggg cgcggggggg ctggcgagct actgtaaact 3541 ttaaagaatt cctgcaagat atttttataa actttt

[0081] SEQ ID NO.' s: 18 and 19 are with reference to: Accession NO. AF533876; 3117 bp; Homo sapiens BR serine/threonine kinase 1 (BRSK2) setting forth:

/translation="MTSTGKDGGAQHAQYVGPYRLEKTLGKGQTGLVKLGVHCVTCQ K VAIKIVNREKLSESVLMKVEREIAILKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGE

LFDYLVKKGRLTPKEARKFFRQI ISALDFCHSHSICHRDLKPENLLLDEKNNIRIADF GMASLQVGDSLLETSCGSPHYACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDD DNLRQLLEKVKRGVFHMPHFIPPDCQSLLRGMSEVDAARRLTLEHIQKHIWYIGGKNE PEPEQPIPRKVQIRSLPSLEDIDPDVLDSMHSLGCFRDRNKLLQDLLSEEENQEKMIY FLLLDRKERYPSQEDEDLPPRNEIDPPRKRVDSPMLNRHGKRRPERKSMEVLSVTDGG SPVPARRAIEMAQHGQRSRSISGASSGLSTSPLSSPRVTPHPSPRGSPLPTPKGTPVH TPKESPAGTPNPTPPSSPSVGGVPWRARLNSIKNSFLGSPRFHRRKLQVPTPEEMSNL TPESSPELAKKSWFGNFISLEKEEQIFVVIKDKPLSSIKADIVHAFLSIPSLSHSVIS

QTSFRAEYKATGGPAVFQKPVKFQVDITYTEGGEAQKENGIYSVTFTLLSGPSRRFK R VVETIQAQLLSTHDPPAAQHLSDTTNCMEMMTGRLSKCGIIPKS"

ORIGIN

1 tgttcggctc agctgcacgg ctcggctcgg ctcggctcgg ctcggctgcg cggccgctga

61 cgggcgtgcg ctgggggcgc ggggcgcggg gcgcgggcct cggcggcggc ggcggcggcg

121 gcggcggaag ccaggtgccc ccgcccgccc tgtcctctcg acgaggcgga ggcgtcgccg

181 cgggccaggc ctcggactgc cgcgtcggag tggacgcggg gggcggcggc gcgggcggac

241 gcgggcggcg cgaagcagcg gggcccgcgg gggcgccccg gccgggtcgg cgcggacggc

301 actcggcgga cgcgggcgga cgctgggcgg cccctccctg cccgcgcgcc cgggcgcccc

361 tggccggcgc tgggccccag agcgatgaca tcgacgggga aggacggcgg cgcgcagcac

421 gcgcagtatg ttgggcccta ccggctggag aagacgctgg gcaaggggca gacaggtctg

481 gtgaagctgg gggttcactg cgtcacctgc cagaaggtgg ccatcaagat cgtcaaccgt

541 gagaagctca gcgagtcggt gctgatgaag gtggagcggg agatcgcgat cctgaagctc 601 attgagcacc cccacgtcct aaagctgcac gacgtttatg aaaacaaaaa atatttgtac 661 ctggtgctag aacacgtgtc aggtggtgag ctcttcgact acctggtgaa gaaggggagg 721 ctgacgccta aggaggctcg gaagttcttc cggcagatca tctctgcgct ggacttctgc 781 cacagccact ccatatgcca cagggatctg aaacctgaaa acctcctgct ggacgagaag 841 aacaacatcc gcatcgcaga ctttggcatg gcgtccctgc aggttggcga cagcctgttg 901 gagaccagct gtgggtcccc ccactacgcc tgccccgagg tgatccgggg ggagaagtat 961 gacggccgga aggcggacgt gtggagctgc ggcgtcatcc tgttcgcctt gctggtgggg 1021 gctctgccct tcgacgatga caacttgcga cagctgctgg agaaggtgaa gcggggcgtg 1081 ttccacatgc cgcactttat cccgcccgac tgccagagtc tgctacgggg catgagcgag 1141 gtggacgccg cacgccgcct cacgctagag cacattcaga aacacatatg gtatataggg 1201 ggcaagaatg agcccgaacc agagcagccc attcctcgca aggtgcagat ccgctcgctg 1261 cccagcctgg aggacatcga ccccgacgtg ctggacagca tgcactcact gggctgcttc 1321 cgagaccgca acaagctgct gcaggacctg ctgtccgagg aggagaacca ggagaagatg 1381 atttacttcc tcctcctgga ccggaaagaa aggtacccga gccaggagga tgaggacctg 1441 cccccccgga acgagataga ccctccccgg aagcgtgtgg actccccgat gctgaaccgg 1501 cacggcaagc ggcggccaga acgcaaatcc atggaggtgc tcagcgtgac ggacggcggc 1561 tccccggtgc ctgcgcggcg ggccattgag atggcccagc acggccagag gtctcggtcc 1621 atcagcggtg cctcctcagg cctttccacc agcccactca gcagcccccg ggtgacccct 1681 cacccctcac caaggggcag tcccctcccc acccccaagg ggacacctgt ccacacgcca 1741 aaggagagcc cggctggcac gcccaacccc acgcccccgt ccagccccag cgtcggaggg 1801 gtgccctgga gggcgcggct caactccatc aagaacagct ttctgggctc accccgcttc 1861 caccgccgaa aactgcaagt tccgacgccg gaggagatgt ccaacctgac accagagtcg 1921 tccccagagc tggcgaagaa gtcctggttt gggaacttca tcagcctgga gaaggaggag 1981 cagatcttcg tggtcatcaa agacaaacct ctgagctcca tcaaggctga catcgtgcac 2041 gccttcctgt cgattcccag tctcagccac agcgtcatct cccaaacgag cttccgggcc 2101 gagtacaagg ccacgggggg gccagccgtg ttccagaagc cggtcaagtt ccaggttgat 2161 atcacctaca cggagggtgg ggaggcgcag aaggagaacg gcatctactc cgtcaccttc 2221 accctgctct caggccccag ccgtcgcttc aagagggtgg tggagaccat ccaggcccag 2281 ctgctgagca cacacgaccc gcctgcggcc cagcacttgt cagacaccac taactgtatg 2341 gaaatgatga cggggcggct ttccaaatgt ggaattatcc cgaaaagtta acatgtcacc 2401 tccacgaggc catcctctgt gaccgaaggc agctgctgcg gacccgccct ccctccgctc 2461 ctgctgttgc tgccgggcag tgaggcccag cccagcgccc cgtccacccc gcggcagctc 2521 ctcgcctcag ctccgcacgg cccgtgggag gaaggccagg ctcgggggag cctcctccag 2581 cccggccgac ccggactccc ggtcacctga cccctcagca agaacagcct gcctggtggc 2641 cttctggggc caggaccccc ggtgggcaac gtagccacag gaacaggccc cgtccaccgc 2701 ctccacgccg cacctggagg cctcctgcag gcccgtgccc cgcctccctg gccgcgccgc 2761 ctccgtgtag tcttggcctc ctcaggctgc ctcccgtcct ctcgtctcac ccgcgcctcc 2821 cttgcctcat ctggggaggc tgtgggctct ggcgctcctc tctggctgag gtggaaacag 2881 agacaccctg cggcaccaga gccttcccag caggccaggc cgctgggctg ggatcagtgt 2941 tatttatttg ccgttttcca atttatggat tctccgcacc tctgttcagg gaagggcggc 3001 ggccacatcc cctgccgtct gcgtgtctca ggcagtgggg gggctggggc cagggcgccc 3061 tctgaggaca gagctggtgg ggcgcggggg ggctggcgag ctactgtaaa ctttaaa

//

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[0082] Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference. Amino acid and nucleic acid sequences for PBKl are shown in appended pages marked "Appendix" which are considered part of this application and

which are incorporated herein by reference in their entirety. Additionally U.S. Patent Application Publications 2003/0092036 and 2005/0125852 are incorporated herein by reference in their entirety.

[0083] The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims. All numerical ranges described herein include all integers and decimal values within the range and are also inclusive of the endpoints.