Ktori, Chariklia; (2004) Investigations of the mechanisms involved in the regulation of class II phosphoinositide 3-kinase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

There is growing evidence that the class-II phosphoinositide (PI) 3-kinases (PI3K) play important roles in regulating cell function with evidence emerging for their role in regulating cellular functions including; clathrin coated vesicle trafficking; nuclear mRNA processing; and the activation of Protein Kinase-B (PKB). The alpha isoform of the class-II PI3K (PI3K-C2α) is activated by a range of growth factors and chemokines, as well as by integrin receptors but the mechanisms responsible for this are poorly understood. As a number of the stimuli known to activate PI3K-C2α are also capable of activating the Protein Kinase C (PKC) and Extracellular Regulated Kinase (ERK) pathways, we investigated whether these could be involved in regulating these aspects of PI3K-C2α biochemistry. Here we demonstrate that phorbol esters such as Phorbol-12-Myristate-13-Acetate (PMA) activate PI3K-C2α in different cell types. Using inhibitors of the ERK and PKC cascades we show that phorbol ester-induced activation of PI3K-C2α is ERK-dependent, but PKC-independent; whereas, the insulin-induced activation of PI3K-C2α is independent of ERK but dependent upon other classes of PI3K. Furthermore, we find that PMA and insulin both induce the phosphorylation of PI3K-C2α as shown by bandshifts. The PMA-induced bandshifts were found to be ERK-and PKC-dependent, whereas the insulin-induced bandshift was dependent upon other classes of PI3K. Our studies provide evidence that phorbol esters induce the production of phosphatidylinositol-3-phosphate (PtdIns-3-P), and this is consistent with PtdIns-3-P being produced by PI3K-C2α. We further show that insulin causes PI3K-C2α to associate with two tyrosine phosphorylated proteins one of 160kDa and another of 106kDa, whereas PMA stimulation causes the association of PI3K-C2α with a tyrosine phosphorylated protein of 120kDa. We show that the 120kDa band contains a member of the FAK and pl30cas family of focal adhesion protein. We also find that the cytokines leptin and Tumour Necrosis Factor alpha (TNFα) induce an activation of PI3K-C2α, and this activation is dependent upon ERK, which is similar to that seen with phorbol esters. Our studies further show that adrenaline and insulin are both involved in the regulation of PI3K-C2α in rat soleus muscle and that cAMP analogues activate PI3K-C2α implying that Protein Kinase A (PKA) can also activate PI3K-C2α. In conclusion we propose that there are different pathways involved in the regulation of PI3K-C2α.

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