Eight human isoforms of phosphoinositide 3-kinases (PI3Ks) exist, but their individual functions remain poorly understood. Here, we show that different human small cell lung carcinoma (SCLC) cell lines overexpress distinct subsets of class I A and II PI3Ks, which results in striking differences in the signalling cascades activated by stem cell factor (SCF). Overexpression of class I A p85/p110a in SCLC cells increased SCF-stimulated protein kinase B (PKB) activation and cell growth, but did not affect extracellular signal-regulated kinase (Erk) or glycogen synthase kinase-3 (GSK-3). This effect was selective, since it was not observed in SCLC cell lines overexpressing p85/p110b or p85/p110d. The SCF receptor associated with both class I A p85 and class II PI3KC2b, and both enzymes contributed to SCFstimulated PKB activity. A dominant-negative PI3KC2b blocked both PKB activation and SCLC cell growth in response to SCF. Together our data provide novel insights into the speci®city and functional signi®cance of PI3K signalling in human cancer. Keywords: cell growth/c-Kit/PI3K/PKB/SCLC
IntroductionThe binding of a wide range of extracellular signals to their speci®c membrane receptors activates PI3K signalling in eukaryotic cells. These enzymes phosphorylate phosphatidylinositol (PI) on the D-3 position of the inositol ring, producing three distinct second messengers, PI(3)P, PI(3,4)P 2 and PI(3,4,5)P 3 (PIP 3 ) (Vanhaesebroeck et al., 2001). The diverse cellular responses regulated by PI3Ks re¯ect the existence of different isoforms of these enzymes with speci®c regulatory mechanisms and functions. The eight PI3Ks identi®ed in various species can be subdivided into three main classes, based on sequence homology and in vitro substrate speci®city (Vanhaesebroeck et al., 2001).Class I A includes three highly homologous isoforms: p110a, p110b and p110d. These three p110s exist as a heterodimeric complex with a p85 regulatory subunit containing two src homology-2 (SH2) domains, which mediates their association with activated growth factor receptors. All the PI3Ks of class I A can phosphorylate PI, PI(4)P and PI(4,5)P 2 in vitro. However, activation of the class I A PI3Ks by receptor tyrosine kinases (RTKs) has been demonstrated to lead mainly to the accumulation of PIP 3 in vivo (Vanhaesebroeck et al., 2001). PIP 3 is the key second messenger in the activation of phosphoinositidedependent kinase-1 (PDK1) (Alessi et al., 1997), which controls various downstream cascades leading to cell growth and survival via protein kinase B (PKB)/Akt (Burgering and Coffer, 1995;Kauffmann-Zeh et al., 1997), ribosomal protein S6 kinase (S6K) (Alessi et al., 1998) and glycogen synthase kinase-3 (GSK-3) (Frame and Cohen, 2001). Accordingly, disruption of the p85a gene in mice impaired B-cell development and proliferation (Fruman et al., 1999).Class II of PI3Ks comprises human PI3KC2a, PI3KC2b and PI3KC2g (Vanhaesebroeck et al., 2001). The hallmarks of class II family members are a substrate speci®city restricted to PI and PI(4)P in vitro, a...