Screening method for ligands with class II P13 kinase...

Details Screening method for ligands with class II P13 kinase... Screening method for ligands with class II P13 kinase...

2002-03-07

2004-08-03

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S252300, C435S320100, C435S325000, C435S419000, C435S254110, C536S023200

Reexamination Certificate

active

06770467

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to a novel class II PI3 kinase, termed PI3K-C2&agr;, and in particular the isolation thereof having regard to its relevant sequence structure and/or biochemical characteristics; means used in the isolation or production thereof; antibodies adapted to bind thereto; and assay kits relating thereto.
Phosphoinositides have been implicated in a variety of cellular processes as diverse as vacuolar protein sorting (1,2), cytoskeletal remodelling (3) and mediating intracellular signalling events through which growth factors, hormones and neurotransmitters exert their physiological effects on cellular activity, proliferation and differentiation (4,5,6).
Recently a family of proteins have been cloned and characterised and shown to be enzymes catalysing the addition of phosphate to inositol. Eukaryotic cells contain a variety of inositol derivatives phosphorylated to different extents. PtdIns(3)P is constitutively present in eukaryotic cells and its levels are constant upon extracellular stimulation. PtdIns(3,4)P
2
and PtdIns(3,4,5)P
3
(7,8) are virtually absent in resting cells but are rapidly induced upon stimulation with a variety of ligands. The enzymes catalysing these reactions are phosphoinositide lipid kinases (hereinafter called PI3 kinases). A brief overview of the current data in relation to PI3 kinases classifies these enzymes into three distinct groups being designated to an individual class by their in vitro substrate specificity, biochemical characteristics and, in examples where a definitive function has been assigned, the nature of the biochemical activity regulated by the specific kinase.
PI3 kinase class 1 polypeptides have a broad spectrum activity, phosphorylating inositol lipids PtdIns, PtdIns(4)P and PtdIns(4, 5)P
2
. Class I kinases are subdivided into Class IA and IB. Class IA polypeptides include p110&agr; (9), p110&bgr; (10) and p110&dgr; (11) which interact physically with the adaptor sub-unit protein p85. Moreover, p110&agr; p100 have a broad distribution in terms of expression pattern. p110&dgr; expression seems to be restricted to white blood cells. Class 1B includes p110&ggr; (12) which functions independently of p85. Although each of these Class 1 kinases catalyse phosphate addition to inositol lipid, the mechanism via which these enzymes are activated and regulated is achieved by different molecular mechanisms.
Class II PI3 kinases have a restricted substrate specificity phosphorylating PtdIns and PtdIns(4)P but not PtdIns(4,5) P
2
. Each of the kinases of this class are characterised by a conserved C2 domain in the carboxyl terminal region of the protein. The presence of conserved motifs within the C2 domain indicates that this region may confer regulation via calcium and/or phospholipid. A comparison of the murine and Drosophila class II kinases mp170 and PI3K
—
68D respectively reveals a high degree of homology in the kinase domain of these proteins. Significant divergence occurs at the amino terminal regions of these polypeptides suggesting that adaptor proteins interacting with these variable domains may regulate kinase activity. Class II PI3 kinases do not interact with p85.
The third class of PI3 kinase, class III PI3 Kinase, is related to the
S.cerevisiae
gene Vps34 (1). This kinase was originally isolated as a gene involved in regulating vesicle mediated membrane-trafficking in yeast. The human homologue of Vps34 is complexed with a ser/thr kinase called Vps15p (14,15). Of the three classes of PI3 kinase this has the most restricted substrate specificity being strictly limited to PtdIns.
SUMMARY OF THE INVENTION
A novel human class II PI3 kinase is herein described and termed human PI3K-C2&agr;. It is characterised as a class II kinase due to the presence of a conserved C2 domain found in murine and Drosophila class II PI3 kinases (FIG.
2
), its apparent lack of a p85 binding site and a substrate specificity limited to PtdIns and PtdIns (4) P (FIG.
4
). The polypeptide is unique in that this is the first human class II kinase to be described. It has significant divergence in the amino terminal region of the protein when compared to the mouse homologue of human PI3K-C2&agr; (16). It is also, surprisingly, the first PI3 kinase to be isolated that has resistance to PI3 kinase inhibitors Wortmannin and LY294002 (FIG.
5
).
The use of selected inhibitors has proved extremely useful in analysing intracellular signal transduction cascades. Inhibitors used at low concentrations probably result in the modification of a single protein's function thereby allowing the dissection of single transduction pathways. A good example of this is the use of Pertussis toxin which is a cell permeant agent (17). The agent undergoes endocytosis into intact cells and results in the ADP-ribosylation of specific GTP-binding or G-proteins. This modification uncouples these G-proteins from their receptors therefore interfering with the cell's response to receptor stimuli. Wortmannin is another cell permeant inhibitor (18,19). It is a fungal metabolite and has been shown to have in vivo anti-inflammatory or immunosuppressive effects in animal models. Wortmannin was first shown to inhibit cellular responses to receptor stimulation in neutrophils. The drug inhibited the respiratory burst induced by ligands such as N-formyl-Met-Leu-Phe,(fMLP),C5a,leukotriene B4 or platelet-activating factors. Importantly, Wortmannin failed to inhibit cellular response to TPA suggesting differential responses to the drug. In particular, the fact that the stimulation of calcium mobilising receptors is resistant to Wortmannin suggests that intracellular signalling initiated by these receptors is controlled by a quite separate kinase cascade.
The identification of PI3 kinases as the target for Wortmannin came from in vitro metabolic labelling of guinea pig neutrophils with
32
p to monitor the uptake of phosphate into phospholipids in the presence of specific kinase inhibitors (20). In control experiments stimulation of fMLP receptors resulted in
32
p labelling of phosphatidic acid and PtdIns(3,4,5)P
3
. The presence of Pertussis toxin had no effect on the phosphorylation of these phospholipids. However, pre-incubation of leucocytes with Wortmannin resulted in inhibition of
32
P incorporation into only PtdIns(3,4,5)P
3
. Since this is the product of PI3 kinase catalysed reactions it seems likely that Wortmannin was specifically targeting PI3 kinase. Subsequently, Wortmannin has been shown to block a number of physiological processes including many insulin stimulated actions that would result in enhanced glucose utilisation (21). Wortmannin has proved to be an effective inhibitor of mammalian PI3 kinases. To date, no human PI3 kinase has been cloned and shown to be resistant to this agent.
The isolation and sequencing of an as yet unidentified human PI3 kinase that has significant homology to previously identified murine and Drosophila class II kinases is described (16,22,23). Comparison of the optimal alignment of these aforementioned proteins shows the human protein to be 32.5% homologous with sequences of the Drosophila PI3K
—
68D and cpk proteins and 90.8% and 90.2% with the murine proteins mp107 and mcpk, FIG.
2
. The carboxyl terminal region of the aforementioned proteins have increased homology due to the presence of a conserved C2 domain. This domain is characterised by the presence of motifs likely to be involved in the modulation of kinase activity by calcium and/or phospholipid.
The amino terminal region of human PI3K-C2&agr; is extended by 176 amino acid residues which are lacking in the murine sequence of mp170. The murine mcpk protein has a 28 amino acid residue deletion in this region that is absent from human PI3K-C2&agr; and a mp170. This divergence in sequence may be explained by the presence of the unique binding sites for adaptor proteins that regulate kinase activity. This is supported by the lack of an apparent p85 binding motif.
Furthermore, immunofluoresence experiments using a monoclonal antibody to the amin

Affiliated with

Also associated with

Rating

Screening method for ligands with class II P13 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 Screening method for ligands with class II P13 kinase..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Screening method for ligands with class II P13 kinase... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3278711