Class II Phosphoinositide 3-Kinases Are Downstream Targets of Activated Polypeptide Growth Factor Receptors

Abstract

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2␣ and PI3K-C2␤ are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2␣ and PI3K-C2␤ represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2␣ and PI3K-C2␤ immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca 2؉ as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2␣ is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2␤ was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2␤ with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2␤ occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2␤ was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3 phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.The binding of polypeptide growth factors to their cell surface receptors triggers the recruitment of numerous molecules to form a localized signaling complex at the plasma membrane. Translocation to the activated receptor from intracellular compartments and conformational and posttranslational modifications all contribute to activate many of the recruited secondary messenger molecules and thus perpetuate the signaling cascade (57). The accumulation of 3Ј phosphoinositides has been observed in numerous cell types following their stimulation with polypeptide growth factors, cytokines, and chemotactic agents (19,25). In quiescent cultures, levels of phosphatidylinositol(3,4)-bisphosphate [PtdIns(3,4)P 2 ] and phosphatidylinositol(3,4,5)-triphosphate [PtdIns(3,4,5)P 3 ] are low but increase rapidly in response to cell stimulation (54). Conseque...

Accelerated protein evolution analysis reveals genes and pathways associated with the evolution of mammalian longevity

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Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

“…Western blotting using anti-PI3K-C2␣ antibodies (Arcaro et al, 2000) demonstrated that PI3K-C2␣ is present in chromaffin cell extracts and enriched in the chromaffin granule fraction (Figure 2). In these samples, the anti-PI3K-C2␣ antibody detected a single band with an apparent molecular weight of 200 kDa, further confirming its specificity.…”

“…In fibroblasts, PI3K-C2␣ has been shown to be enriched on the TGN and clathrin-coated vesicles (Arcaro et al, 2000). On the TGN, PtdIns3P has been shown to be required for the formation of constitutive transport vesicles (Jones and Howell, 1997).…”

“…We used affinity purified polyclonal antibody against PI3K-C2␣ ( Arcaro et al, 2000), anti-hGH (Sigma), anti-Syt I (Matthew et al, 1981), anti-P18 (Wendler et al, 2001), anti-EEA1 (a kind gift from H.T. McMahon) and anti-clathrin heavy chain (Transduction Laboratories).…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…In support of this hypothesis, there is emerging evidence that class II PI3K is a significant producer of PtdIns3P, and class II PI3K holds a wide spectrum of functions ranging from cell signaling, to cell migration and vesicle trafficking. [161][162][163][164][165][166] One hint implying a role of class II PI3K in autophagy was provided by the studies on cell corpse clearance by phagocytosis in C. elegans. 167,168 In this process, autophagy is required for phagosome maturation and clearance of apoptotic cells; most importantly, class II PI3K was identified as a complementary kinase producing PtdIns3P on phagosomes, indispensable for the degradation of cell corpses in C. elegans.…”