Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhi bitory proper-ties of its N-terminal and C-terminal domains but can be acti vated approximately 40-fold in vitro by polyunsaturated fatty acids. To ide ntify residues involved in regulating PP5 activity, we performed scanning m utagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and dele tion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain project s away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different f rom that involved in heat shock protein 90 binding. We also observed a 10-f old elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-st imulated activity. These two effects were accounted for by two distinct ami no acid deletions: deleting the four C-terminal residues (496-499) of PP5 h ad no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, b ut deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimula ted activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5. our data suggest that Asn-491, near its C-terminus, may also be involved in th is process.