Structural and Biochemical Evaluation of the Interaction of the Phosphatidylinositol 3-Kinase p85a Src Homology 2 Domains with Phosphoinositides and Inositol Polyphosphates

Macromolecular Biochemistr

Molecular cloning, cDNA sequence, and chromosomal localization of the human phosphatidylinositol 3-kinase p110 alpha (PIK3CA) gene.

Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme comprising a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that binds to tyrosine phosphopeptide sites linked directly or indirectly to receptors serving diverse signal functions. Knowledge of the structure and function of PI 3-kinase was greatly advanced by the purification, cDNA cloning, and subsequent expression of the bovine enzyme. Here the cloning of the cDNA for the human p110 alpha subunit of PI 3-kinase (PIK3CA), encoding a protein 99% identical to the bovine p110, and of its gene in YAC is described. The chromosomal localization of the gene for PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the gene in 3q26.3

Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells.

BACKGROUND: As phosphoinositides can serve as signalling molecules within cells, the enzymes responsible for their synthesis and cleavage are likely to be involved in the transduction of signals from the cell surface through the cytoplasm. The precise role of the phosphoinositide 3-kinase that has been cloned from mammalian cells is not known, but it has been implicated in receptor-stimulated mitogenesis, glucose uptake and membrane ruffling. The enzyme can use phosphatidylinositol (PtdIns), PtdIns 4-phosphate and PtdIns (4,5)-bisphosphate as substrates in vitro, but it seems to phosphorylate PtdIns (4,5)-bisphosphate preferentially in vivo. The VPS34 gene product of yeast, by contrast, is a phosphoinositide 3-kinase homologue implicated in vacuolar protein sorting that apparently utilizes only PtdIns as a substrate. The significance of this difference in lipid-substrate preference and its relationship to the functions of the two phosphoinositide kinases is unknown. RESULTS: We have characterized a distinct PtdIns-specific phosphoinositide 3-kinase activity in mammalian cells. Unlike the previously identified, broad-specificity mammalian phosphoinositide kinase, this enzyme is resistant to the drug wortmannin and uses only PtdIns as a substrate in vitro; it therefore has the capacity to generate PtdIns 3-phosphate specifically. The newly characterized enzyme, which was purified by chromatography from cytosol, has biochemical and pharmacological characteristics distinct from those of the broad-specificity enzyme. CONCLUSIONS: The enzyme we have characterized may serve to generate PtdIns 3-phosphate for fundamentally different roles in the cell from those of PtdIns (3,4)-bisphosphate and/or PtdIns (3,4,5)-trisphosphate. Furthermore, the functions of the VSP34 gene product, which may not be relevant to the broad-specificity mammalian phosphoinositide 3-kinase, may be related to those of the enzyme we describe

A human phosphatidylinositol 3-kinase complex related to yeast Vps34p-Vps15p protein sorting system

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

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Republication: Targeting PI3KC2β Impairs Proliferation and Survival in Acute Leukemia, Brain Tumours and Neuroendocrine Tumours.

Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. The expression pattern and functions of the class II PI3KC2β isoform were investigated in a panel of tumour samples and cell lines. Overexpression of PI3KC2β was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2β or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2β also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. Together, these data show that PI3KC2β contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies