Abstract
Pleckstrin is a substrate for protein kinase C in activated platelets that contains at its N and C termini two of the pleckstrin homology (PH) domains that have been proposed to mediate protein-protein and protein-lipid interactions. We have recently shown that pleckstrin can inhibit agonist-induced phosphoinositide hydrolysis and that this inhibition requires an intact N-terminal PH domain (residues 6 to 99). In the present studies, we have identified the sites of phosphorylation in pleckstrin and examined their contribution to pleckstrin function. In human platelets activated with thrombin or phorbol esters, and in COS-1 cells expressing pleckstrin, a combination of phosphopeptide analysis and site-directed mutagenesis shows that three residues in the intervening sequence between the two pleckstrin PH domains become phosphorylated: Ser113, Thr114, and Ser117. Replacing all three of these sites with glycine decreased phosphorylation by > 90% and reduced pleckstrin's ability to inhibit phosphoinositide hydrolysis by as much as 80%. Replacing the phosphorylation sites with alanine residues had a similar effect, while substitution with aspartate, glutamate, or lysine residues produced pleckstrin variants that were fully active even in the absence of phosphorylation. These results suggest that phosphorylation enhances pleckstrin's activity by introducing a cluster of charges into a region adjacent to, but not within, the N-terminal PH domain. This may have an allosteric effect on the N-terminal PH domain, regulating its interaction with other molecules necessary for the inhibition of phosphoinositide hydrolysis.
Highlights
Proteins that are important in signal transduction often contain discrete domains that mediate protein-protein interactions
The results show that: 1) pleckstrin is variably phosphorylated on a cluster of residues located near, but not within, the N-terminal pleckstrin homology (PH) domain, 2) elimination of any one of these sites does not alter the overall phosphorylation of the molecule, presumably because of compensatory increases in phosphorylation at the other sites, 3) phosphorylation of pleckstrin is required for maximal inhibition of phosphoinositide hydrolysis, and 4) the effects of phosphorylation may be due largely to the introduction of charged residues into the region between the two PH domains
Replacement of Phosphorylation Sites with Charged Residues—Since the pleckstrin expressed in the transfected COS-1 cells becomes phosphorylated when the cells are stimulated with thrombin, we examined whether replacing the sites of phosphorylation with charged, rather than neutral, amino acids would mimic the effects of phosphorylation
Summary
Proteins that are important in signal transduction often contain discrete domains that mediate protein-protein interactions. The inhibition of phosphoinositide hydrolysis required an intact N-terminal PH domain and was additive with that observed when mock-transfected cells were preincubated briefly with PMA, suggesting that it is independent of the phosphorylation of receptors, G-proteins, and phospholipase C known to occur under the same conditions (19 –22).
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