Publications by Year: 2010

Class IA phosphoinositide 3-kinase (PI3K) p110 catalytic subunits are activated upon Src homology 2 domain-mediated binding of their p85 regulatory subunits to tyrosine-phosphorylated pYXXM motifs in receptor tyrosine kinases (RTKs) or adaptor proteins. The PI3K pathway is activated by phosphate and tensin homolog (PTEN) loss in most prostate cancers (PCa), but the contribution of upstream RTKs that may be targeted therapeutically has not been assessed. Immunoblotting of p85-associated proteins in serum-starved PTEN-deficient LNCaP and C4-2 PCa cells showed a small set of discrete tyrosine-phosphorylated proteins, but these proteins were not recognized by an anti-pYXXM motif antibody and were not found in PTEN-deficient PC3 PCa cells. LC/MS/MS using label-free proteomics and immunoblotting showed that p85 was associated primarily with p110beta and p110delta. An interaction with ErbB3 was also detected but was independent of ErbB3 tyrosine phosphorylation and was not required for basal PI3K activity. Basal tyrosine phosphorylation of p110beta and p110delta could be blocked by c-Src inhibitors, but this did not suppress PI3K activity, which was similarly independent of Ras. Basal PI3K activity was mediated by p110beta in PC3 cells and by both p110beta and p110delta in LNCaP cells, whereas p110alpha was required for PI3K activation in response to RTK stimulation by heregulin-beta1. These findings show that basal PI3K activity in PTEN-deficient PCa cells is RTK-independent and can be mediated by p110beta and p110delta. Increased p110beta expression in PCa may be required for RTK-independent PI3K pathway activation in adult prostate epithelium with genetic or epigenetic PTEN down-regulation.

This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term iNKT derives from the fact that a large fraction of murine NKT cells recognize the MHC class I-like CD1d protein, are CD4+ or CD4-CD8- (double negative), and use an identical "invariant" TCRalpha chain, which is generated by precise Valpha14 and Jalpha281 (now renamed Jalpha18) rearrangements with either no N-region diversity or subsequent trimming to nearly identical amino-acid sequence (hence, 'iNKT'). Basic Protocol 1 and Alternate Protocol 1 use multi-color FACS analysis to identify and quantitate rare iNKT cells from human samples. Basic Protocol 2 describes iNKT cell purification. Alternate Protocol 2 describes a method for high-speed FACS sorting of iNKT cells. Alternate Protocol 3 employs a cell sorting approach to isolate iNKT cell clones. A Support Protocol for secondary stimulation and rapid expansion of iNKT cells is also included. Basic Protocol 3 explains functional analysis of iNKT.