3-phosphoinositide-dependent protein kinase

Details 3-phosphoinositide-dependent protein kinase 3-phosphoinositide-dependent protein kinase

1997-10-03

2004-05-11

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S015000, C435S069100, C530S350000, C424S094500

Reexamination Certificate

active

06734001

ABSTRACT:

The present invention relates to enzymes, to polynucleotides encoding enzymes and to uses of enzymes and polynucleotides.
Protein kinase B (PKB) [1] or RAC protein kinase [2] is the cellular homologue of a viral oncogene v-Akt [3] and has therefore also been termed c-Akt. The current interest in PKB stems firstly from the discovery that it is activated rapidly in response to insulin and growth factors and that the activation is prevented by inhibitors of phosphoinositide (PI) 3-kinase [4-6]; secondly, from the finding that PKB isoforms are overexpressed in a significant percentage of ovarian, pancreatic [7, 8] and breast cancer cells [2].
PKB appears to mediate the insulin-induced inhibition of glycogen synthase kinase-3 (GSK3) in L6 myotubes which is thought to underlie, at least in part, the insulin-induced dephosphorylation and activation of glycogen synthase [9] and protein synthesis initiation factor eIF2 [10] that contribute to the stimulation of glycogen and protein synthesis by insulin. However PKB is likely to have other physiological substrates and, in transfection based experiments, has been shown to activate p70 S6 kinase [5], to stimulate the translocation of the glucose transporter GLUT4 to the plasma membrane and enhance glucose uptake into 3T3-L1 adipocytes [11], and to mediate the IGF1-induced survival of neurones [12] and fibroblasts [13] against apoptosis.
A critical question concerns the mechanism by which PI 3-kinase triggers the activation of PKB. The activation of PKB is accompanied by its phosphorylation [5,14] and we recently showed that activation by insulin or IGF1 resulted from its phosphorylation at Thr-308 and Ser-473 [15].
Moreover, the insulin or IGF1 induced phosphorylation of both residues was abolished by wortmannin, an inhibitor of PI 3-kinase [15]. We believe that the protein kinases which phosphorylate PKB at Thr-308 and Ser-473 might themselves be activated by phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3), the product of the PI 3-kinase reaction. In the work presented here, we demonstrate that this is indeed the case, and we describe the purification and characterisation of a 3-phosphoinositide-dependent protein kinase (PDK1) which activates PKB and we disclose polynucleotides which encode PDK1 and uses for PDK1 and said polynucleotides.
A first aspect of the invention provides a substantially pure 3-phosphoinositide dependent protein kinase that phosphorylates and activates protein kinase B&agr;.
By “substantially pure” we mean that the 3-phosphoinositide-dependent protein kinase is substantially free of other proteins. Thus, we include any composition that includes at least 30% of the protein content by weight as the said 3-phosphoinositide-dependent protein kinase, preferably at least 50%, more preferably at least 70%, still more preferably at least 90% and most preferably at least 95% of the protein content is the said 3-phosphoinositide-dependent protein kinase.
Thus, the invention also includes compositions comprising the 3-phosphoinositide-dependent protein kinase and a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5% of the composition by weight.
The invention also includes the substantially pure said 3-phosphoinositide-endent protein kinase when combined with other components ex vivo, said other components not being all of the components found in the cell in which said protein kinase is found.
It is preferred that the substantially pure 3-phosphoinositide-dependent protein kinase is a substantially pure phosphatidyl-3,4-5-trisphosphate-dependent protein kinase or a substantially pure phosphatidyl-3,4-bisphosphate-dependent protein kinase.
By “phosphorylates protein kinase B&agr;” we include the meaning that the 3-phosphoinositide-dependent protein kinase is able to transfer a phosphate group from ATP to an acceptor group of protein kinase B&agr;. Preferably, the acceptor group is Thr-308.
By “protein kinase B&agr;” we include any protein kinase B&agr; or any suitable derivative or fragment thereof or fusion of protein kinase B&agr; or derivative, or fragment thereof. For example, it is particularly preferred that the protein kinase B&agr; is a fusion between glutathione-S-transferase and protein kinase B&agr; as described in Example 1 (GST-PKB&agr;; see also reference 27).
It is preferred that the PKB&agr; is a human PKB&agr;. It should be appreciated that the said 3-phosphoinositide-dependent protein kinase from one species or tissue can phosphorylate and activate PKB&agr; from another species or tissue.
By “activates protein kinase B&agr;” we include the meaning that upon phosphorylation by the said 3-phosphoinositide-dependent protein kinase the activity of protein kinase B&agr; to a given substrate increases by at least ten-fold compared to the protein kinase B&agr; which has not been so phosphorylated, preferably by at least 20-fold and more preferably by at least 30-fold. Suitably, the activity of protein kinase B&agr; is measured using the synthetic peptide RPRAATF (SEQ ID NO: 9).
By “3-phosphoinositide-dependent protein kinase” we include the meaning that the protein kinase is substantially inactive at activating PKB&agr; in the absence of a suitable 3-phosphoinositide (or a compound that mimics the effect of a 3-phosphoinositide). In particular, the said protein kinase has at least ten-fold increased activity towards protein kinase B&agr; in the presence of a 3-phosphoinositide compared to the activity in the absence of said 3-phosphoinositide, preferably at least 100-fold, more preferably at least 1000-fold, and still more preferably at least 10,000-fold.
It will be appreciated that the 3-phosphoinositide-dependent protein kinase may be activated by mimics of 3-phosphoinositide as described in more detail below.
Preferably, the activation of PKB&agr; by the 3-phosphoinositide-dependent protein kinase is substantially accelerated by the D-enantiomer of sn-1-stearoyl-2-arachidonyl phosphatidylinositol 3,4,5-trisphosphate but is not substantially accelerated by the L-enantiomer of the said phosphatidylinositol 3,4,5-trisphosphate.
Preferably, the 3-phosphoinositide-dependent protein kinase is substantially activated by the D-enantiomer of sn-1,2-dipalmitoyl phosphatidylinositol 3,4,5-trisphosphate or sn-1,2-dipalmitoyl phosphatidylinositol 3,4-bisphosphate but is not substantially activated by the L-enantiomers of the said phosphatidylinositol phosphates.
Preferably, the 3-phosphinositide-dependent protein kinase is not substantially activated by phosphatidylinositol 3,5-bisphosphate or phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3-phosphate or inositol 1,3,4,5-tetrakisphosphate.
Thus, particularly with reference to FIG.
6
and the rabbit skeletal muscle PDK1 of Example 1, the following 3-phosphoinositides have been found to activate the said 3-phosphoinositide-dependent kinase (in order of level of activation; most effective first):
1. Lipid 5: Racemic sn-1,2-dilinoleoyl PtdIns(3,4,5)P
3
.
2. (Equal) Lipid 2: D-enantiomer of sn-1-stearoyl-2-arachidonyl PtdIns(3,4,5)P
3
.
2. (Equal) Lipid 3: D-enantiomer of sn-2-arachidonyl-3-stearoyl PtdIns(3,4,5)P
3
.
4. (Equal) Lipid 6: sn-1,2 di-palmitoyl PtdIns(3,4,5)P
3
.
4. Equal Lipid 7: sn-1,2 di-palmitoyl PtdIns(3,4)P
2
.
The following phospholipids cause no significant activation, at least in relation to rabbit skeletal muscle PDK1:
6. Lipid 2: L-enantiomer of sn-1-stearoyl-2-arachidonyl PtdIns(3,4,5)P
3
.
7. Lipid 4: L-enantiomer of sn-2-arachidonyl-3-stearoyl PtdIns(3,4,5)P
3
.
8. Lipid 9: PtdIns(4,5)P
2
.
9. Lipid 8: sn-1,2 di-palmitoyl PtdIns(3,5)P
2
.
10. Lipid 11: sn-1,2 di-palmitoyl PtdIns-3P.
11. Lipid 10: PtdIns 4P.
12. IP
4
: Ins(1,3,4,5P
4
.
We have found that the huma

Affiliated with

Also associated with

Rating

3-phosphoinositide-dependent protein kinase does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with 3-phosphoinositide-dependent protein kinase, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and 3-phosphoinositide-dependent protein kinase will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3194229