P T Hawkins

The Gβγ Sensitivity of a PI3K Is Dependent upon a Tightly Associated Adaptor, p101

Two highly similar, PtdIns(4,5)P2-selective, G beta gamma-activated PI3Ks were purified from pig neutrophil cytosol. Both were heterodimers, were composed of a 101 kDa protein and either a 120 kDa or a 117 kDa catalytic subunit, and were activated greater than 100-fold by G beta gammas. Peptide sequence-based oligonucleotide probes were used to clone cDNAs for the p120 and p101 species. The cDNA of p120 is highly related to p110 gamma, while the cDNA of p101 is not substantially related to anything in current databases. The proteins were expressed in and purified from insect and mammalian cells. They bound tightly to one another, both in vivo and in vitro, and in so doing, p101 amplified the effect of G beta gammas on the PI3K activity of p120 from less than 2-fold to greater than 100-fold.

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.