Geering, B.; (2006) Regulation of class IA phosphoinositide 3-kinase signalling enzymes by post-translational modifications, protein interactions and absolute protein expression levels. Doctoral thesis , University of London.

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Abstract

Phosphoinositide 3-kinases (PI3Ks) are signal transduction proteins that regulate a wide range of cellular processes. A key subset of PI3Ks are the so-called class IA enzymes consisting of a p85 regulatory and p110 catalytic subunit. While the biological activities of PI3Ks have been investigated in detail, the biochemical mechanism of their regulation is underexplored. We therefore strived to investigate post-translational modifications and absolute protein levels of class IA PI3Ks using electrophoresis and mass spectrometry (MS). We thereby focused on endogenous PI3K subunits and physiological stimulation. Modifications including amino acid substitutions, phosphorylation and hydroxylation were detected in the regulatory subunit of unstimulated cells. While modifications in p85 were unaltered following cellular stimulation, dynamic changes in the tyrosine phosphorylation of the catalytic subunit p1105 were detected upon B cell stimulation. p1105 tyrosine phosphorylation does not appear to be critical for intrinsic PI3K lipid kinase activity, but might have a role in the interaction of p110S with known and novel potential PI3K partners identified by MS. However, identification of modified amino acid residues by MS turned out to be challenging, also because p110 proteins could not be resolved by 2D-gel electrophoresis. An important question in the PI3K field is whether class IA subunits can exist as monomers. It has been reported that p85 is present in excess over p110 and suggested that monomeric p85 has a negative role in the regulation of PI3K activity. Our analysis of class IA PI3K protein expression, amongst other by affinity and ion exchange chromatography, does not support this notion. In summary, our work has revealed dynamic changes in the post-translational modification of the p110 (but not p85) and their interaction with partners. The ratio of p110 to p85, however, does not seem to be as dynamic, with equal amounts of p85 to p110 under all conditions investigated.

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