Date of Award
Doctor of Philosophy (PhD)
Calvin B. Harley
Pleckstrin is the major substrate of PKC in activated platelets, therefore a role for pleckstrin in mediating platelet responses such as secretion has been widely hypothesized. Previous cloning of the pleckstrin gene indicated that it encoded a unique protein sequence containing no obvious structural, catalytic or otherwise functional motifs, apart from several candidate PKC phosphorylation sites and homology between the Nand C terminal domains.
The role of pleckstrin in mediating cytoskeletal changes that occur during platelet activation and secretion was investigated based on published data implicating pleckstrin in directly inhibiting actin polymerization in its unphosphorylated fonn. There were no significant effects on the kinetics of actin polymerization in vitro using recombinant pleckstrio or pleckstrin purified from platelets. In accordance with this observation, overexpression of pleckstrin in COS cells did not appear to affect the subcellular distribution or ftlament length of actin compared to uninfected cells. Also, pleckstrin did not significantly co-localize with actin prior to or after exposure to phorbol 12-myristate 13- acetate (PMA) concentrations known to induce maximal phosphorylation of pleckstrin. It was therefore concluded that pleckstrio does not playa direct role in regulating actin polymerization.
Based on limited sequence homology to dynamin (which is involved in endocytosis), I postulated that pleckstrio might regulate vesicle motility, a key requirement for platelet degranulation. Pleckstrin immunofluorescently labelled in transfected COS cells did not appreciably co-localize with either ER or Golgi structures, the precursors of secretory vesicles, or with microtubules, however some punctate fluorescence was observed that co-localized with the ER. Immunfluorescent detection of pleckstrin in peripheral blood cells indicated that pleckstrin is most highly abundant in platelets, moderately expressed in polymorphonuclear cells and absent or lowly expressed in lymphocytes.
Pleckstrin's prevailing characteristic, its phosphorylation, was examined in greater detail. Wild-type recombinant pleckstrin overexpressed in COS cells displayed a 5 fold stimulation of ³²P incorporation in response to PMA treatment. To exclude the possiblity of phosphorylation by kinases other than PKC, the effect of staurosporine on inhibiting pleckslrio phosphorylation was examined. Staurosporine inhibited pleckstrin phosphorylation in the heterologous COS cell system with an IC₅₀ = 10 nM. similar to that reported to inhibit pleckstrin phosphorylation in the platelet. Additionally. identical phosphopeptide maps were generated of wild-type pleckslrio isolated from HL-60, HEL or transfected COS cells indicating that pleckstrin expressed recombinantly in COS cells was by all known measures functionally competent. However, a pleckstrin mutant deleted for 42 residues including the most probable phosphorylation sites was de-regulated for phosphorylation, displaying a high basal level of phosphorylation, no response to PMA, and increased association with cytoskeletal or membraneous components.
To precisely map the phosphorylation sites of pleckstrin. 8 serine or threonine residues were converted to alanine using site-directed mutagenesis. Two of these mutants, S113A and SI17 A, consistently displayed a lower level of PMA-induced hosphorylation compared to the wild-type protein. Tryptic phosphopeptide maps of three pleckstrin mutants, S1l3A, T1l4A and S1l7A indicated that phosphorylation of pleckstrin occurs almost exclusively on SI13 and S117; these sites accounted for 46 and 52% respectively of the incorporated phosphate. A minor, variable amount of phosphorylation on TI14 was observed. These phosphorylation sites were confirmed by phosphopeptide mapping synthetic peptides corresponding to residues 108-120 of pleckstrio, and by performing phosphoamino acid analysis on the tryptic phosphopeptides generated in these maps.
Lastly, overexpression of each of the phosphorylation site mutants of pleckstrin in COS cells indicated that one of them, S 113A, might possess a dominant activity that perturbs membrane/cytoskeletal structures. This mutant induced large, semi-circular ruffles or "lobes" in transfected COS cells in two independent experiments.
In conclusion, the function of pleckstrin is still entirely unknown. It is quite clear however that pleckstrin is an excellent substrate for PKC. Phosphorylation of pleckstrin likely affects its function, a hypothesis supported in this work by the unusual phenotype of COS cells overexpressing the Sll3A mutant.
Craig, Karen Leigh, "Characterization and phosphorylation site mapping of human pleckstrin" (1996). Open Access Dissertations and Theses. Paper 3422.