Insights into enzyme-catalysed phosphoryl transfer from studies of MAP kinases

Phosphoryl transfer is one of the most common biological reactions, and enzymes catalysing this are capable of producing huge rate enhancements that has led to great interest in their catalytic mechanism. This thesis focuses on p38α and MEK6, protein kinases belonging to one of the MAP kinase signalling pathways in eukaryotic cells, in which MEK6 activates p38α by dual phosphorylation of tyrosine and threonine in its activation loop. Protein phosphatases which dephosphorylate the same residues play a role in regulating these pathways. Chapter 3 demonstrates and investigates a novel phosphatase activity of MEK6 towards p38α. This phosphatase activity is found to be dependent on activation loop phosphorylation, is metal cation dependent and requires the general base aspartate. Together these results indicate that the kinase and phosphatase activities utilise the same active site. Whilst this dual kinase/phosphatase activity is unexpected, similarities in the mechanism of protein kinases and PPP/PPM phosphatases suggest that the active site is naturally equipped to catalyse both reactions. Chapter 4 concerns studies on other protein kinases, either serine/threonine specific or dual specificity, to try to determine how widely phosphatase activity is preserved amongst members of the kinome. The results of these studies suggest that phosphatase activity is a specific feature of the MAP2K’s which are archetypal dual specificity kinases. Chapter 5 focuses on unexpected kinase activity, firstly investigating the ability of MEK6 and other protein kinases to transfer the β-phosphate of ADP (rather than ATP γ-phosphate) to substrate. The findings show that these kinases can use ADP as a phosphoryl donor, which may be functionally significant in conditions of limited ATP supply. In addition this chapter describes the unexpected formation of a transition-state analog complex between MEK6 and p38α lacking both activation sites, implying a nonspecific kinase activity of MEK6.