Effects of MPSD and Ala-MPSD on the phosphorylation of MARCKS induced by PKC activation.

Platelets were labeled with [³²P]P_i, permeabilized with digitonin, incubated with 100 nM PMA in the absence or presence of 10 μM of either MPSD or Ala-MPSD, and subsequently stimulated as described in the legend to Figure 5. At the end of the stimulation periods, heat-stable platelet extracts were prepared, and their proteins were separated by SDS-PAGE as indicated in “Materials and methods.” Proteins were then electrotransferred to nitrocellulose membranes; these were exposed to hyperfilm, and the autoradiographies thus obtained were scanned as indicated in “Materials and methods.” Panel A shows the autoradiography of an experiment carried out to test the effect of MPSD on protein phosphorylation, and panel B shows a similar experiment performed with Ala-MPSD. Double arrows in A and B show the position of MARCKS, and single arrowheads indicate the position of myosin light chain (MLC). The open triangles in A indicate the position of proteins p25, p31, p50, and p66. The asterisk in A indicates the position of peptide MPSD, which was less phosphorylated in the absence (basal PKC activity) than in the presence of PMA (basal + stimulated PKC activity). Note the absence of a phosphorylated band when the experiment was performed with Ala-MPSD (B). On the right side in both panels, scannings of the autoradiographies are shown. The numbers beside the MARCKS and MLC peaks are arbitrary units obtained from computer integration of peak areas. Similar results were obtained in 3 other experiments (see Figure9B).