| Summary: | Phosphoinositide 3-kinases (PI3Ks) are a family of signal transduction enzymes which generate lipid second messengers (1) and control a wide variety of cellular processes such as growth, proliferation, survival, differentiation, intracellular traffic, cytoskeletal organisation and cell migration (1-7) Mammals have 8 isoforms of PI3K, most of which have poorly defined individual roles in cells and in the organism. Deregulated PI3K signalling has been implicated in cancer, inflammation and diabetes, and PI3Ks are being pursued as new therapeutic targets by the pharmaceutical industry. Therapeutic intervention with PI3K will almost certainly have to be targeted at individual isoforms, given the risk that global alteration of PI3K signalling will be deleterious to the organism. This work focuses on the pi 108 isoform of PI3K. pi 105 belongs to the class IA subset of PI3Ks which signal downstream of tyrosine kinase receptors and Ras. These PI3Ks are heterodimers that consist of a pi 10 catalytic subunit and a regulatory subunit. Mammals have genes for 3 catalytic subunits, called pi 10a, pi 10(3 and pi 105. Whereas pi 10a and pllOp show a broad tissue distribution, pi 105 is more restricted, with the highest levels in leukocytes (8) There is mounting evidence for a non-redundant function of these pi 10 isoforms (7,9). Studies using mice have implicated pi 105 in immune signalling, making it an interesting new target for anti-inflammatory therapies. More recent work (7) also indicates a role for pi 105 in cancer. This thesis studies the mechanism of tissue specific distribution of pi 105 at the mRNA and protein level. Using Real-Time reverse transcriptase PCR we have demonstrated that regulation of pi 105 expression occurs at the level of transcription. Using two different approaches, 5' RACE (Rapid Amplification of cDNA Ends) and reverse transcriptase PCR, we have also discovered different transcription start sites in the pi 105 mRNA, leading to three discrete mRNAs which contain distinct 5' untranslated exons. In a further stage of the work, we have found evidence that differential usage of these 5' untranslated exons correlates with the distinct tissue distribution of the pi 105 protein. Our findings imply that there are at least 3 distinct promoters for the pi 105 gene. Efforts to delineate the promoter elements using reporter assays and deletion analysis have given ambiguous results, and have not lead to the desired insight into the regulation of the pi 105 promoters and their regulation in different tissues.
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