Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-03-10
2001-10-09
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S320100, C435S325000, C435S252300, C536S023200
Reexamination Certificate
active
06300111
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to molecules that participate in growth factor receptor signaling and cell proliferation. More particularly, the invention relates to a constitutively active phosphatidylinositol 3′-kinase, methods for its production, and diagnostic and therapeutic uses of the activated kinase and its derivatives.
2. Description of the Background Art
Growth factors play a role in embryonic development, cancer, atherosclerosis and the responses of tissues to injury. Growth factors are involved in several normal developmental processes as well as in pathological conditions.
One particular growth factor, platelet-derived growth factor (PDGF) is a major mitogen for mesenchymal cells, smooth muscle cells and glial cells. The binding of PDGF to its receptor (PDGF-R) triggers a diverse group of early cellular responses including activation of tyrosine kinases, stimulation of phosphatidylinositol turnover, alterations in ion fluxes, activation of phospholipase A2, changes in cell shape and the enhanced expression of a group of early response genes including the c-fos and c-myc proto-oncogenes.
In vivo, PDGF is stored in the &agr; granules of blood platelets and does not circulate freely in blood. During blood clotting and platelet adhesion, the granules are released, often at sites of injured blood vessels, implicating PDGF in the repair of blood vessels. PDGF also stimulates migration of arterial smooth muscle cells from the medial to the intimal layer of the artery where they then proliferate as an early response to injury. Thus, activation of the PDGF-R receptor is involved in wound healing, in atherosclerosis, and in stimulating genes associated with cancerous transformation of cells, particularly c-myc and c-fos.
Because disregulation of the cellular processes involved in cell growth can have disastrous effects, it is important to understand and gain control over these processes. This requires identifying the participants in the signaling events that lead to mitogenesis and elucidating their mechanism of function. To this end, several cytoplasmic signalling molecules that mediate the mitogenic action of growth factor receptors, in particular, the PDGFR, have been identified and studied. One of these molecules is phosphatidylinositol 3-kinase.
Phosphatidylinositol 3-kinase is one of many activities stimulated by growth factors. Phosphatidylinositol 3-kinase is known to be involved in the regulation of cell growth and oncogenic transformation (Cantley et al.,
Cell,
64:1657 (1993)). The enzyme is found associated with receptor protein tyrosine kinases such as PDGF-R-&bgr;, CSF-1 receptor, Insulin receptor and IGF-1 receptor as well as non-receptor tyrosine kinase oncogenes, e.g., src, gag-abl and fyn. Studies on mutants of platelet-derived growth factor (PDGF) receptor have shown that phosphatidylinositol 3-kinase is a key mediator of PDGF-mediated mitogenic signaling (Fantl et al.,
Cell,
69:413 (1992); Valius et al., ibid., 73:321 (1993)). PDGF-R mutants that are unable to bind phosphatidylinositol 3-kinase are also unable to induce a mitogenic response after growth factor stimulation and unable to activate p21
c-ras
(ras). These data suggested that phosphatidylinositol 3-kinase acts upstream of ras in PDGF-stimulated signaling.
Until now, it has not been possible to examine the action of phosphatidylinositol 3-kinase directly, because the only known way of activating phosphatidylinositol 3-kinase involved the use of tyrosine kinases that are known to bind, phosphorylate and localize phosphatidylinositol 3-kinase. The use of receptor mutants to study signaling pathways has disadvantages, since several signaling molecules might share the same binding site on the receptor and it cannot be excluded that other signaling pathways are also affected by the mutation. Therefore it would be of importance to have a phosphatidylinositol 3-kinase molecule that functions independent of prior growth factor stimulation. The present invention satisfies this and other needs.
Phosphatidylinositol 3-kinase is a heterodimer consisting of a 110 kD catalytic subunit, p110 (the sequence for mouse p110 is hereby designated SEQ ID NO:11), and an 85 kD regulatory subunit, p85 (the sequence for mouse p85 is hereby designated SEQ ID NO:12). Upon growth factor receptor stimulation, the wild-type phosphatidylinositol 3-kinase is activated and can phosphorylate phosphatidylinositol at the 3′ position of the inositol ring. Phosphatidylinositol 3-phosphates are candidate second messenger molecules. The catalytic subunit of phosphatidylinositol 3-kinase, p110, exhibits enzymatic activity in mammalian cells only when bound to the p85 subunit or to 102 amino acids of the p85 inter-SH2 (iSH2) region (Klippel et al.,
Mol. Cell. Biol.,
14:2675-2685 (1994)). This iSH2 fragment bound to a region at the extreme N-terminus of p110 (Klippel et al., ibid., 14:2685 (1994)). Attempts to reconstitute a functional phosphatidylinositol 3-kinase in a cell-free system by mixing p85 expressed in
E. coli
with in vitro-translated p110 or by mixing the two subunits which had been separately expressed in COS cells or
E. coli,
have been less than successful.
SUMMARY OF THE INVENTION
The present invention constructs a constitutively active form of phosphatidylinositol 3-kinase that could be expressed in cells. This active phosphatidylinositol 3-kinase is advantageous in that it allows the activities of the enzyme to be studied directly and obviates the problems described. This constitutively active phosphatidylinositol 3-kinase that acts uncoupled from growth factor stimulation is useful in elucidating how phosphatidylinositol 3-kinase triggers cellular responses involved in the regulation of cell growth and provides novel approaches to the regulation or modulation of cell proliferation and therapeutic intervention in cancer.
In the present invention, a constitutively active phosphatidylinositol 3-kinase is produced by combining just the elements of p85 and p110 which are essential for activity, into a single molecule. This approach offers several advantages including that only one expression construct needs to be transfected into each cell. In addition, a single iSH2-p110 fusion molecule ensures a 1:1 stoichiometry of the active elements from each subunit.
A constitutively active p110 mutant, p110*, was constructed by attaching the p85 iSH2 region to the p110 N-terminal domain. Both domains were connected via a glycine-kinker that serves as a hinge region thereby giving the iSH2 domain more flexibility to interact with the p110 N-terminal domain. When expressed in mammalian cells p110* had reproducibly higher specific phosphatidylinositol 3-kinase activity than wildtype p110 coexpressed with iSH2. p110 also has protein-kinase activity. As with phosphatidylinositol 3-kinase activity, the protein-kinase activity is dependent upon the association of p110 with iSH2.
The invention provides a method of producing a constitutively active phosphatidylinositol 3-kinase by fusing the iSH2 domain sequences of the p85 subunit to the amino terminus of the p110 catalytic subunit using a linker, preferably glycine. p110-p85iSH2 expression constructs and methods of preparing them, methods of expressing the fusion enzyme in cells, and isolation and purification of the constitutively active enzyme are disclosed.
This constitutively active phosphatidylinositol 3-kinase has various uses. One aspect of the invention relates to the use of p110* as a convenient and abundant source of active enzyme to generate inositol phosphate products in vitro. These products such as PI 3′4′-P
2
, PI 3′,4′,5′-P
3
and PI 3′-P are commercially useful as reagents, particularly in studying lipid metabolism and signal transduction. The invention also provides kits containing the consitutively active phosphatidylinositol 3-kinase and other necessary reagents as well as instructions for use of the kinase in preparing inositol phosphate products.
The invention
Klippel Anke
Williams Lewis T.
Prouty Rebecca E.
The Regents of the University of California
Townsend & Townsend & Crew LLP
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