Compositions and methods for identifying PKB kinase inhibitors

Details Compositions and methods for identifying PKB kinase inhibitors Compositions and methods for identifying PKB kinase inhibitors

1998-09-17

2004-01-27

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S069100, C435S252300, C435S320100, C435S015000, C536S023200

Reexamination Certificate

active

06682920

ABSTRACT:

FIELD OF THE INVENTION
This invention is in the field of molecular biology and involves the identification of protein kinase B kinases, and their medical applications.
BACKGROUND OF THE INVENTION
Insulin, and other growth factors, trigger the activation of phosphatidylinositol (PI) 3-kinase, the enzyme which converts PI 4,5 bisphosphate (PIP2) to the putative second messenger PI 3,4,5 trisphosphate (PIP3). There are multiple forms of PI 3-kinase, and they arc all able to phosphorylate the D-3 position of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns4-P) and phosphatidylinositol4,5-bisphosphate (PtdIns4,5-P2) to produce phosphatidylinositol-3-phosphate (PtdIns-3-P), phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol-3,4,5-triphosphate (PtdIns-3,4,5-P3, or PIP3), respectively.
Protein kinase B (PKB) is in the signal transduction pathway of PIP3, and lies downstream of PI 3-kinase. See, Franke et al., (1995) Cell, vol. 81, pages 727-736. For example, activation of PKB by insulin or growth factors is prevented if the cells are preincubated with inhibitors of PI 3-kinase, the best known being Wortmannin or LY 294002, or by overexpression of a dominant negative mutant of PI 3-kinase. See, Burgering, B M. and Coffer, P. J. (1995) Nature, vol. 376, pages 599-602. Further, mutation of the tyrosine residues in the PDGF receptor that when phosphorylated bind to PI 3-kinase also prevent the activation of PKB&agr;, an isoform of PKB. Recent reports have shown the PKB is itself activated by another kinase also downstream of PIP3. See, Alessi, D. R. et al., Curr. Biol. vol. 7, 261 (1997). This kinase, termed PKB kinase, or phosphatidylinositide (PtdIns) 3-kinase (PDK1), requires PIP3 for activation. See, Stokoe, D., et al (1997) Science, vol. 277, pages 567-570.
PKB is a key enzyme in the PIP3 pathway, and is involved in regulating cell growth. It has been implicated in certain human cancers; for instance, it is known to be amplified in a percentage of ovarian carcinomas, breast carcinomas, and pancreatic carcinomas. See, Bellacosa, A. et al. (1995) Int. J. Cancer 64, pages 280-285, and Cheng, J. Q. et al. (1996) Proc. Natl. Acad. Sci. U.S.A. vol. 93, 3636-3641. The amplification of the enzyme affords tumor cells a mechanism to circumvent apoptosis. Thus, it will be appreciated that drugs that inhibit PKB activity will be beneficial for the treatment of diseases involving unwanted cell growth, including cancer. One way to achieve this end is to develop assays that identify, such.
SUMMARY OF THE INVENTION
A first object of the invention is a description of PKB kinases, methods and compositions for purifying and expressing the kinases, and cDNA sequences that encode them.
A second object of the invention is a description of the activation of PKB kinases by PtdIns (3,4,5)P
3
to effect the phosphorylation of PKB.
A third object of the invention is a description of compositions and methods for identifying compounds that have prophylatic or therapeutic benefit for treating diseases involving unwanted cell growth, including cancer.
These and other objects of the invention will become apparent to a skilled practitioner of this art upon a full disclosure of the invention.


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Frech, M., et al., 1997, The Journal of Biological Chemistry, vol. 272, “High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity”, pp. 8574-8481.*
Shaw, M., et al., 1997, FEBS Letters, vol. 416, “Further evidence that the inhibition of glycogen synthase kinase 3beta is mediated by PDK1/PKB-induced phosphorylation of Ser-9 and not by dephosphorylation of Tyr-216”, pp. 307-311.*
Alessi D.R. et al. “Characterization of a 3-phosphoinositide-dependent . . . ” Current Biology, V. 7, No. 4, Mar. 1997, pp. 261-269.
Alessi D.R. et al. “3-phosphoinositide-dependent protein kinase-1 (PDK1) . . . ” Current Biology, V. 7, No. 10, Sep. 1997, pp. 776-789.
Stokoe et al., “Dual role of phosphatidyl-3,4,5-triphosphate in the . . . ” Science, V. 277, Jul. 1997, pp. 567-570.
P. Cohen et al., “PDK1, one of the missing links in insulin signal transduction?” FEBS Letters, V. 410, No. 1, Jun. 1997, pp. 3-10/.
L. Stephens et al., “Protein kinase B kinases that mediate phosphatidylinositol . . . ” Science, V. 279, Jan. 1998, pp. 710-714.

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