Class I and Class III P13 kinases in endocytosis, integrin signaling, and nutrient sensing

Abstract

PI3-kinases are a family of lipid kinases that phosphorylate the 3-hydroxyl group on phosphoinositides. The p110beta isoform is unique among the Class I PI3-kinases in that it can be activated both by RTKs and by GPCRs. In addition, only p110beta binds to the early endosomal small GTPase Rab5, suggesting a role for p110beta in endocytic trafficking. To study the regulation of p110beta by Rab5, we identified point mutations within the helical domain of p110beta that inhibit Rab5 binding, yet have no effect on p110beta kinase activity. Cells expressing Rab5-uncoupled mutant p110beta showed a minor defect in EGF uptake but were defective for EGF-stimulated fluid phase uptake, macropinocytosis, and formation of circular dorsal ruffles. In addition, expression of Rab5-uncoupled p110beta led to enhanced chemokinesis in MEFs, by an integrin-dependent mechanism. A distinct PI 3-kinase, the Class III enzyme Vps34, plays important roles in endocytosis and autophagy, and has been demonstrated to regulate nutrient sensing and activation of the mammalian Target of Rapamycin Complex 1 (mTORC1) in cultured cells. We collaborated with Dr. Bart Vanhaesebroeck to study mice that were heterozygous for a kinase-dead allele of Vps34. The mice exhibited increased glucose tolerance and insulin sensitivity, yet had increased fat accumulation. However, no differences between amino acid-stimulated mTORC1 signaling in the heterozygote and wild type mice was observed. We also studied Vps34 signaling in Zebrafish embryos. Vps34 morphants exhibited heart edema, yolk defects, a curved body axis, a melanocyte migration defect, a phagocytosis defect as well as a partial inhibition of autophagy. These studies indicate that Zebrafish is a useful in vivo vertebrate model for studying the signaling by Vps34. The work presented in this thesis has produced useful tools for further investigation into p110beta and Vps34 signaling.