GSK3 polypeptides

Details GSK3 polypeptides GSK3 polypeptides

2001-07-25

2002-10-15

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S015000, C530S350000, C536S023200

Reexamination Certificate

active

06465231

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention provides materials and methods relating to identification and optimization of selective inhibitors of glycogen synthase kinase 3 (GSK3), and also relates to methods of treating a condition mediated by GSK3 activity. Such conditions include Alzheimer's disease, type 2 diabetes, and inflammation.
2. Description of the Related Art
Glycogen synthase kinase 3 (GSK3) is a proline-directed serine/threonine kinase originally identified as an activity that phosphorylates glycogen synthase as described in Woodgett
Trends Biochem Sci.
16:177-181 (1991). The role in glucose metabolism has been elaborated recently in Summers et al.,
J. Biol. Chem.
274:17934-17940 (1999). GSK3 consists of two isoforms, &agr; and &bgr;, and is constitutively active in resting cells, inhibiting glycogen synthase by direct phosphorylation. Upon insulin activation, GSK3 is inactivated, thereby allowing the activation of glycogen synthase and possibly other insulin-dependent events. GSK3 is inactivated by other growth factors or hormones that, like insulin, signal through receptor tyrosine kinases. Examples of such signaling molecules include IGF-1 and EGF as described in Saito et al.,
Biochem. J.
303:27-31 (1994), Welsh et al.,
Biochem. J.
294:625-629 (1993), and Cross et al.,
Biochem. J.
303:21-26 (1994). GSK3 has been shown to phosphorylate &bgr;-catenin as described in Peifer et al.,
Develop. Biol.
66:543-56 (1994). Other activities of GSK3 in a biological context include GSK3's ability to phosphorylate tau protein in vitro as described in Mandelkow and Mandelkow,
Trends in Biochem. Sci.
18:480-83 (1993), Mulot et al.,
Febs Lett
349: 359-64 (1994), and Lovestone et al.,
Curr. Biol.
4:1077-86 (1995), and in tissue culture cells as described in Latimer et al.,
Febs Lett
365:42-6 (1995). Selective inhibition of GSK3/may be useful to treat or inhibit disorders mediated by GSK3 activity.
There is a need in the art for compositions and molecules that bind to or interact with GSK3, thereby mediating GSK3 activity. The invention meets this need by providing crystallizable GSK3 polypeptides useful for design and optimization of GSK3 inhibitors.
BRIEF SUMMARY OF THE INVENTION
The invention provides GSK3&bgr; molecules with N- and C-terminal truncations, wherein the molecules are capable of crystallization.
The invention further provides GSK3&bgr; molecules truncated at amino acid R
344
, R
354
, T
364
, A
374
, and I
384
.
The invention provides a polypeptide consisting essentially of SEQ ID NO:2 or SEQ ID NO:3, polynucleotides encoding these polypeptides, and vectors comprising these polynucleotides.
The invention still further provides GSK3&bgr; molecules wherein translation of the molecule begins at G
34
, T
39
, P
44
, D
49
or V
54
.
The invention also provides GSK3&agr; molecules with N- and C-terminal truncations, wherein the molecules are capable of crystallization.
The invention further provides a GSK3&agr; molecule wherein translation of the molecule begins at S
97
and ends at S
447
, polynucleotides encoding this polypeptide, and vectors comprising these polynucleotides.
The invention further provides a method of identifying a GSK3 polypeptide capable of crystallization, comprising: (a) providing a truncated GSK3 polypeptide; (b) testing the polypeptide for formation of crystals.
The invention also provides GSK3 polypeptides capable of interacting with inhibitors of GSK3.
The invention further provides a method of identifying an enzymatically active GSK3 polypeptide, comprising: (a) providing a truncated GSK3 polypeptide; (b) contacting the polypeptide with a substrate of GSK3; and (c) measuring the kinase activity of the polypeptide after contacting the polypeptide with the substrate, wherein the polypeptide is active if it shows >0.01×the activity of the full-length enzyme and preferably >0.1×the activity of the full-length enzyme.


REFERENCES:
patent: 6057117 (2000-05-01), Harrison et al.
patent: WO 99/65897 (1999-12-01), None
US 6,057,286, 5/2000, Harrison et al. (withdrawn)
Stambolic et al., Swiss-Prot accession No. P49841, Oct. 1, 1996.*
Cross et al., “The inhibition of glycogen synthase kinase-3 by insulin of insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf,”Biochemical Journal 303(Part 1):21-26, Oct. 1, 1994.
Latimer et al., “Stimulation of MAP kinase by v-raf transformation of fibroblasts fails to induce hyperphosphorylation of transfected tau,”FEBS Letters 356(1):42-46, May 22, 1995.
Lovestone et al., “Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells,”Current Biology 4(12):1077-1086, Dec. 1, 1994.
Mandelkow and Mandelkow “Tau as a marker for Alzheimer's disease,”TIBS 18(12):480-483, Dec. 1993.
Mulot et al., “PHF-tau from Alzheimer's brain comprises four species on SDS-PAGE which can be mimicked by in vitro phosphorylation of human brain tau by glycogen synthase kinase 3-&bgr;,”FEBS Letters 349(3):359-364, Aug. 8, 1994.
Peifer et al., “Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase,”Developmental Biology 166(2):543-556, Dec. 1994.
Saito et al., “The mechanism by which epidermal growth factor inhibits glycogen synthase kinase 3 in A431 cells,”Biochemical Journal 303(Part 1):27-31 Oct. 1, 1994.
Summers et al., “The role of glycogen synthase kinase 3&bgr; in insulin-stimulated glucose metabolism,”Journal of Biological Chemistry 274(25):17934-17940, Jun. 18, 1999.
Welsh and Proud, “Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B,”Biochemical Journal 294(Part 3):625-629, Aug. 15, 1993.
Woodgett, “A common denominator linking glycogen metabolism, nuclear oncogenes and development,”TIBS 16(5):177-181, May 1991.
Jancarik and Kim, “Sparse Matrix Sampling: A Screening Method for Crystallization of Proteins,”J. of Applied Crystallography 24(4):409-411, 1991.

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