Lipid kinase

Details Lipid kinase Lipid kinase

1998-12-01

2002-11-19

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S006120, C435S252300, C435S320100, C435S183000, C536S023200

Reexamination Certificate

active

06482623

ABSTRACT:

The invention relates to a novel lipid kinase which is part of the PI3 Kinase (P13K) family and more specifically the invention relates to various aspects of the novel lipid kinase particularly, but not exclusively, to an identification of expression of said kinase with a view to diagnosing or predicting motility or invasion of cells such as metastasis of cancer cells; and also agents for interfering with said expression or inhibiting said kinase with a view to enhancing or reducing or preventing said motility or invasion so as to enhance or restrict, respectively the movement of selected cells.
An overview of the PI3 kinase family of enzymes is given in our co-pending Patent Application WO93/21328. Briefly, this class of enzymes shows phosphoinositide (hereinafter referred to after as PI) 3-kinase activity. Following major advances in our knowledge of cell signal transduction and cell second messenger systems it is known that the PI3Ks have a major role to play in regulating cell function. Indeed, it is known that PI3Ks are members of a growing number of potential signalling proteins which associate with protein-tyrosine kinases activated either by ligand stimulation or as a consequence of cell transformation. Once thus associated they provide an important complex in the cell signalling pathway and thus direct events towards a given conclusion.
PI3 kinases catalyse the addition of phosphate to the 3′-OH position of the inositol ring of inositol lipids generating phosphatidyl inositol monophosphate, phosphatidyl inositol diphosphate and phosphatidyl inositol triphosphate (Whitman et al, 1988, Stephens et al 1989 and 1991). A family of PI3 kinase enzymes has now been identified in organisms as diverse as plants, slime molds, yeast, fruit flies and mammals (Zvelebil et al, 1996).
It is conceivable that different PI3 kinases are responsible for the generation of the different 3′-phosphorylated inositol lipids in vivo. Three classes of PI3 kinase can be discriminated on the basis of their in vitro lipid substrates specificity. Enzymes of a first class have a broad substrate specificity and phosphorylate PtdIns, PtdIns(4)P and PtdIns(4,5)P
2
. Class I PI3 kinases include mammalian p110&agr;, p110&bgr; and p110&ggr; (Hiles et al, 1192; Hu et al, 1993; Stephens et al, 1994; Stoyanov et al, 1995).
P110&agr; and p110&bgr; are closely related PI3 kinases which interact with p85 adaptor proteins and with GTP-bound Ras.
Two 85 kDa subunits, p85&agr; and p85&bgr;, have been cloned (Otsu et al, 1992). These molecules contain an N-terminal src homology-3 (SH3) domain, a breakpoint cluster (bcr) homology region flanked by two proline-rich regions and two src homology-2 (SH2) domains. Shortened p85 proteins, generated by alternative splicing from the p85&agr; gene or encoded by genes different from those of p85&agr;/&bgr;, all lack the SH3 domain and the bcr region, which seem to be replaced by a unique short N-terminus (Pons et al, 1995; Inukai et al, 1996; Antonetti et al, 1996). The SH2 domains, present in all p85 molecules, provide the heterodimeric p85/p110 PI3Ks with the capacity to interact with phosphorylated tyrosine residues on a variety of receptors and other cellular proteins. In contrast to p110&agr; and &bgr;, p110&ggr; does not interact with p85 but instead associates with a p101 adaptor protein (Stephens et al, 1996). P110&ggr; activity is stimulated by G-protein subunits.
PI3Ks of a second class contains enzymes which, at least in vitro, phosphorylate PtdIns and PtdIns(4)P but not PtdIns(4, 5)P
2
(MacDougall et al, 1995; Virbasius et al, 1996, Molz et al, 1996). These PI3Ks all contain a C2 domain at their C-terminus. The in vivo role of these class II PI3Ks is unknown.
A third class of PI3K has a substrate specificity restricted to PtdIns. These PI3Ks are homologous to yeast Vps34p which is involved in trafficking of newly formed proteins from the Golgi apparatus to the vacuole in yeast, the equivalent of the mammalian lysosome (Stack et al, 1995). Both yeast and mammalian Vps34p occur in a complex with Vps15p, a 150 kDa protein serine/threonine kinase (Stack et al, 1995; Volinia et al, 1995; Panaretou et al, submitted for publication).
PtdIns(3)P is constitutively present in cells and its levels are largely unaltered upon extracellular stimulation. In contrast, PtdIns(3, 4)P
2
and PtdIns(3, 4, 5)P
3
are almost absent in quiescent cells but are produced rapidly upon stimulation by a variety of growth factors, suggesting a likely function as second messengers (Stephens et al, 1993). The role of PI3Ks and their phosphorylated lipids in cellular physiology is just beginning to be understood. These lipids may fulfill a dual role: apart from exerting physical, charge-mediated effects on the curvature of the lipid bilayer, they also have the capacity to interact with specific binding proteins and modulate their localisation and/or activity. Amongst the potential targets for these lipids are protein kinases such as protein kinase C isoforms, protein kinase N/Rho-activated kinases and Akt/RAC/protein kinase B (Toker et al, 1994; Palmer et al, 1995; Burgering and Coffer, 1995; Franke et al, 1995; James et al, 1996; Klippel et al, 1996). Akt/RAC/protein kinase B is likely to be upstream of targets such as p70 S6 kinase and glycogen synthase kinase-3 (Chung et al, 1994; Cross et al, 1995). PI3Ks also affect the activity of small GTP-binding proteins such as Rac and Rab5, possibly by regulating nucleotide exchange (Hawkins et al, 1995; Li et al, 1996). Ultimately, the combination of these actions can result in cytoskeletal rearrangements, DNA synthesis/mitogenesis, cell survival and differentiation (Vanhaesebroeck et al, 1996).
We describe herein a mammalian novel Class I PI3 Kinase which we have termed p110&dgr;. This novel PI3 Kinase typifies the Class I PI3 Kinase family in that it binds p85&agr;, p85&bgr; and p85&ggr;. In addition, it also binds GTP-ras but, like p110&agr;, shows no binding of rho and rac. It also shares the same GTP-broad phosphoinositide lipid substrate specificity of p110&agr; and p110&bgr;, and it also shows protein kinase activity and has a similar drug sensitivity to p110&agr;.
However, it is characterised by its selective tissue distribution. In contrast to p100&agr; and p110&bgr; which seem to be ubiquitously expressed, p110&dgr; expression is particularly high in white blood cell populations i.e. spleen, thymus and especially peripheral blood leucocytes. In addition to this observation we have also found that p110&dgr; is expressed in most melanomas, but not in any melanocytes, the normal cell counterpart of melanomas. Given the natural distribution of p110&dgr; in tissues which are known to exhibit motility or invasion and also the expression of p110&dgr; in cancer cells we consider that p110&dgr; has a role to play in cell motility or invasion and thus the expression of this lipid kinase in cancer cells can explain the metastatic behaviour of cancer cells.
A further novel feature of p110&dgr; is its ability to autophosphorylate in a MN
2
+-dependent manner. Indeed, we have shown that autophosphorylation tends to hinder the lipid kinase activity of the protein. In addition, p110&dgr; contains distinct potential protein:protein interaction modules including a proline-rich region (see
FIG. 1
, position 292-311, wherein 8 out of 20 amino acids are proline) and a basic region leucine zipper (bZIP) like domain (Ing et al., 1994 and Hirai et al., 1996). Such biochemical and structural differences between p85-binding PI3 kinases indicate that they may fulfill distinct functional roles and/or be differentially regulated in vivo.
We disclose herein a nucleic acid molecule, of human origin, and corresponding amino acid sequence data relating to p110&dgr;. Using this information it is possible to determine the expression of p110&dgr; in various tissue types and in particular to determine the expression of same in cancer tissue with a view to diagnosing the motility or invasiveness of such tissue and thus predicting the potential for secondary tum

Affiliated with

Also associated with

Rating

Lipid 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 Lipid kinase, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Lipid kinase will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2984443