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Sequential activation of individual PKC isozymes in integrin-mediated muscle cell spreading: a role for MARCKS in an integrin signaling pathway

¹ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
² Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA
³ GRECC and Neurology Service, Veterans Affairs Palo Alto Heath Care System, Palo Alto, CA 94304, USA

View larger version (71K): [in a new window]   Fig. 1. Morphological and biochemical changes associated with cell attachment and spreading of skeletal muscle cells plated on FN. (A) Myoblasts were plated on FN and photographed at various times after plating. The time in minutes after plating is indicated. The panels show characteristic morphological changes of attachment and spreading, including bleb formation (5 minutes, white arrow), membrane ruffling (5 and 10 minutes, closed arrow) and circumferential lamella (15 minutes, arrowhead). (B) Actin stress fiber formation and FAK localization were determined in myoblasts at different time points after the cells were plated on FN. The upper panels show the development of stress-fiber formation by staining the cells with fluorescently labeled phalloidin. The lower panels show the change in FAK localization from a predominantly diffuse cytosolic localization at 15 minutes to a more focal adhesion site localization (arrows) at later time points. (C) FAK phosphorylation was determined as a function of time after plating on FN. At each time point, the cells were harvested in RIPA buffer. Phosphorylation of FAK was determined by immunoblot analysis using an antiphosphotyrosine antibody. A duplicate blot was probed with an anti-FAK antibody (lower panel) to confirm equal loading of FAK protein in each lane.

View larger version (34K): [in a new window]   Fig. 2. PKC isozyme expression and activation in muscle cells. (A) PKC isozyme expression was determined by western blot analysis in mouse myoblasts. Myoblasts were grown for 2 days then lysed in RIPA buffer. 80 µg of proteins were chromatographed on a 7.5% SDS gel then probed with antibodies specific for individual PKC isozymes. The lanes containing protein from myoblast cultures are labeled `Mb'. Adjacent lanes include positive controls for each isozyme. `B' refers to mouse brain extract (20 µg/lane), which is known to highly express all the isozymes except for PKC and was therefore used as a positive control. For PKC, extract of skeletal muscle tissue [`Mf' (myofibers)] was used (80 µg/lane). (B) PKC isozyme activity increases upon integrin binding and activation. PKC isozymes were immunoprecipitated from total cell extracts of myoblasts plated on FN for different lengths of time, and the activity of each isozyme was determined by the level of 32P incorporation into histone III-S. These results are normalized to the activity at time zero and to the amount of PKC isozymes immunoprecipitated from each sample determined by western blot analysis. These results represent the mean±s.d. from eight separate experiments. (C) 5-expressing and 5-deficient myoblasts were plated on FN for various times. PKC isozymes were immunoprecipitated as described in A, and phosphorylated histone was analyzed on 10% SDS-polyacrylamide gel. A representative autoradiogram is shown. As in A, PKC isozyme activity increases transiently in 5-expressing myoblasts (5(+)) plated on FN. By contrast, there was no increase in PKC isozyme activity in the 5-deficient myoblasts (5-/-) on FN. The protein levels of each isozyme in the two cell populations were indistinguishable by western blot analysis.

View larger version (23K): [in a new window]   Fig. 3. Inhibition of immunoprecipitated kinase activity by a PKC-specific inhibitor. (A) PKC was immunoprecipitated from myoblasts with or without PMA treatment. After immunoprecipitation with an antibody specific for PKC, in vitro kinase assays were carried out in the presence and absence of the PKC inhibitor, chelerythrine (2 µM), using either histone or MBP as a substrate. The phospho-proteins were loaded on a 10% or 12% SDS-polyacrylamide gel then transferred to nitrocellulose followed by autoradiography to assess histone phosphorylation (upper row) or MBP phosphorylation (middle row). The blots were probed with an anti-PKC antibody to confirm equal amounts of PKC protein in each sample (lower row). (B) The incorporation of 32P into histone or MBP from experiments such as that shown in A was quantified. These results presented are averaged from two separate experiments and demonstrate the marked induction of PKC activity by PMA that is maintained after immunoprecipitation and inhibited by chelerythrine.

View larger version (109K): [in a new window]   Fig. 4. PKC isozyme localization in myoblasts plated on FN. Myoblasts were plated on FN for various times, methanol/acetone fixed and stained for individual PKC isozymes. This figure shows characteristic patterns of localization of , and PKC as the cells attach and spread. These results were observed in more than 90% of the cells. PKC is localized at focal adhesion sites (arrow) after 15 minutes on FN. After 15 minutes on FN, PKC revealed a Golgi-like staining (arrow), whereas after 1 hour, PKC showed a punctate staining pattern at the cell periphery (arrow). After 15 minutes, PKC was detected in the nucleus and in perinuclear regions (arrow), and became localized diffusely in the cytosol as well as in the nucleus after 1 hour on FN.

View larger version (38K): [in a new window]   Fig. 5. 5-deficient cell spreading induced by activation of specific PKC isozyme. (A) 5-deficient cells were plated on FN for 30 minutes in the presence or absence of PMA (3 nM), , or peptide activators (1 µM, labeled by the up arrow) or all three peptide activators together. In the absence of any activators, the cells do not attach. PMA treatment promotes rapid attachment and spreading. The activation of , or PKC all promote cell attachment, and and PKC activation promote cell spreading. All three activators together are nearly as effective as PMA. (B) 5-deficient cells treated as in A were assessed for FAK phosphorylation after 30 minutes on FN using an anti-phosphotyrosine antibody. FAK phosphorylation increased in the presence of the PKC activators in parallel with the effect on cell spreading shown in A. A duplicate blot was probed with an anti-FAK antibody to confirm equal loading (lower panel). FAK phosphorylation was quantified to calculate the percentage of activation (shown below each lane), with control levels being defined as no activation and PMA treatment defined as maximal activation. (C) 5-deficient cell spreading induced by , and PKC activators is inhibited by the corresponding specific inhibitors. 5-deficient cells were treated with individual PKC isozyme activators in the presence or absence of isozyme-specific inhibitors. FAK phosphorylation was determined by western blot analysis, and the level of phosphorylation was quantified. The experiment was repeated three times with similar results, and the results of a representative experiment are shown. The data were calculated as percentages of maximum activation obtained after 3 nM PMA treatment.

View larger version (18K): [in a new window]   Fig. 6. PKC is required for cell spreading and FAK phosphorylation in 5-expressing and 5-deficient myoblasts. 5-expressing (5(+)) and 5-deficient (5-/-) cells were plated on FN in the presence or absence of 3 nM PMA, , or PKC inhibitors (downward arrows), or PKC inhibitors in the presence of PMA (3 nM). FAK phosphorylation was determined after 30 minutes. The PKC inhibitor blocks FAK phosphorylation both in 5-expressing cells and in 5-deficient cells activated by PMA, whereas inhibitors of PKC and PKC had little effect. For the loading control, the level of FAK protein was determined by probing duplicate blots with an anti-FAK antibody.

View larger version (74K): [in a new window]   Fig. 7. Actin reorganization in 5-expressing cells plated on FN. Actin localization and stress-fiber formation were observed in 5-expressing cells plated on FN. Paraformaldehyde-fixed cells were stained for actin and stress fibers (F-actin) as indicated in the Materials and Methods. At early time points (15 minutes), actin was found at focal adhesion sites (arrow). With time, focal contacts were distributed uniformly across the cell surface, and fine stress fibers were found around the nucleus and at the periphery of the cell.

View larger version (75K): [in a new window]   Fig. 8. MARCKS localization and translocation in 5-expressing cells plated on FN. (A) MARCKS is localized to focal adhesion sites during the initial phases of muscle cell attachment and spreading. 5-expressing cells were plated on FN, and cells were fixed and co-stained for MARCKS and the focal adhesion protein paxillin. The cells shown here were fixed 30 minutes after plating and show colocalization of MARCKS and paxillin, demonstrating the localization of MARCKS at focal adhesion sites (arrow). (B) MARCKS translocates from the membrane to the cytosol during muscle cell spreading. 5-expressing cells were plated on FN for various times prior to fixation and immunocytochemical assessment of MARCKS localization. MARCKS localization to focal adhesion sites (arrows in each panel) is most prominent at early time points, decreasing in intensity as the cells spread. With time, MARCKS becomes more diffusely distributed in the cytosol. (C) MARCKS translocation is mediated by integrin activation. To confirm the immunocytochemical translocation in B and to assess the role of integrin activation in the process, 5-expressing and 5-deficient cells were plated on FN, and MARCKS translocation was assessed by cellular fractionation. In 5-expressing cells, MARCKS is initially localized predominantly in the membrane compartment, consistent with the localization seen in B. With time, MARCKS translocates to the cytosolic fraction such that by 90 minutes, nearly all of the protein is in this compartment. By contrast, there is almost no translocation of MARCKS from the membrane to the cytosol in 5-deficient cells plated on FN.

View larger version (44K): [in a new window]   Fig. 9. MARCKS is essential for muscle cell spreading. 5-expressing cells were transfected with a MARCKS antisense vector or a vector control, and individual clones are selected for analysis of MARCKS expression and cell spreading. (A) MARCKS expression in transfected clones. Three clones (02, 042 and 011) transfected with the MARCKS-antisense cDNA showed reduced levels of MARCKS protein. A representative clone (`cont') transfected with control vector showed normal levels of MARCKS protein expression. Individual clones were photographed to illustrate the relationship between MARCKS protein expression and cell spreading. The clone transfected with the control vector (empty vector) showed normal cell spreading on FN. By contrast, each of the clones expressing the MARCKS antisense vector showed reduced cell spreading, and the inhibition of cell spreading correlated directly with the extent of reduction of MARCKS protein expression (A). MARCKS protein was undetectable by western blot analysis in clone 02, and this clone showed the most dramatic inhibition of cell spreading. Clone 02 was plated on FN with or without pretreatment with PMA (100 nM). Even activation of PKC by PMA was unable to promote spreading of this clone in which MARCKS protein was undetectable (A).

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