Abstract
Src homology 3 (SH3) domains have been recently shown to bind to proline-rich sequences contained in 3BP1, 3BP2, and SOS. In a recent study we demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) associates with the Fyn SH3 domain. Here we show that p85, the regulatory subunit of PI 3-kinase, binds directly to the SH3 domains of Abl, Lck, Fyn, and p85 itself. An examination of p85 amino acid sequence revealed two proline-rich sequences in its N-terminal region similar to those present in 3BP1, 3BP2, and SOS. To test whether these sequences mediate the association of p85 with SH3 domains two peptides with amino acid composition corresponding to the p85 alpha proline-rich sequences were synthesized and used in competition assays. Both peptides worked equally well in inhibiting the binding of PI 3-kinase activity and p85 alpha to Fyn SH3 domain, whereas a control peptide had no effect. These results indicate that, as in 3BP1 and SOS, the proline-rich sequences in p85 mediate its interaction with SH3 domains. These results also suggest that the SH3 domain of p85 may "self-associate" with the proline-rich motifs of the same subunit as part of the PI 3-kinase regulatory mechanism.
Highlights
These domains habveeen implicatedin regulating examination of p86 amino acid sequence revealed two cytoskeletal functions, because they have beenidentified in proline-rich sequences in its N-terminal region similar several proteinsassociated with the cytoskeleton in both yeast to those present in 3BP1,3BP2,SOanSd
The PI 3-Kinase p85 Subunit Contains ltvo SH3-bindiSnigtes and is mediated by its SH2 domains as in the caseof its association to activated receptor-tyrosine kinases and one that is phosphorylation-independent, in which p85 associates withthe SH3 domains of upstream molecules. Both interactions may occur in parallel in the caseof activated cytosolic-tyrosine kinases thatcontain SH2 and SH3domains
Examination of the p85 amino acid sequence revealed the presence of two proline-rich motifs highly homologous to the ones identifiedin 3BP1,3BP2, anSdOS
Summary
Division of Signal via its regulatory subuni(tp). Examination of the p85 amino. These results suggest that SH3-containing proteins phosphate. Antibodies against the C-terminal SH2 domainof p85a were washed three times (5 min) with TBST(TBS,0.2%Tween 20) and obtained from Transduction (Lexington,KY). Preparation of CelLl ysates-HPB-ALL cellswere pelleted and the beads incubated with SF9lp85a cell lysates were harvested and washed twice with ice-cold phosphate-buffered saline. The lysates were cleared by centrifugation at 15,000 rpm for 10 min figures These conditions weredetermined by time course studies that followed by passage through 0.2-pm filters. Protein concentration was revealed that preincubating the GST fusion protein beads with the determined (Bio-Rad micro-assay),and the lysates were used at a peptides forlongerperiods of time or a t room temperature didnot concentration of 2 rng/dsample.
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