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Myosin II heavy chain isoforms are phosphorylated in an EGF-dependent manner

involvement of protein kinase C

Ravid Straussman^*, Liron Even^* and Shoshana Ravid

Department of Biochemistry, Hadassah Medical School The Hebrew University, Jerusalem 91120, Israel
^* These authors contributed equally to this paper
Author for correspondence (e-mail: ravid{at}md2.huji.ac.il )

View larger version (22K): [in a new window]   Fig. 1. Analysis of MHC-A and MHC-B expression in TSU-pr1. Immunoblots of TSU-pr1 cell extracts immunoprecipitated with antibodies against either MHC-A or MHC-B, or both, were electrophoresed as described (Kelley et al., 1996). Extracts of RBL and COS cells electrophoresed in 7% SDS-PAGE. The proteins were transferred to nitrocellulose membranes and were probed with an antibody specific to either MHC-A or MHC-B, or with both.

View larger version (14K): [in a new window]   Fig. 2. EGF-induced increases in MHC-A and MHC-B phosphorylation of TSU-pr1 cells. Cells were incubated in the presence of 32P orthophosphate and stimulated with 7 ng ml-1 EGF. Aliquots of TSU-pr1 were removed at different times before and after stimulation with EGF and subjected to immunoprecipitation as described in Materials and Methods. The immunoprecipitated MHC-A and MHC-B were analyzed on 7% SDS-PAGE gels and using a PhosphorImager (Fuji). Relative phosphorylation of MHC-A and MHC-B was determined by dividing the values obtained with the PhosphorImager by the values obtained by scanning of the Coomassie-Blue-stained gels, as described in Materials and Methods. The results are the average of three to six experiments.

View larger version (73K): [in a new window]   Fig. 3. EGF-dependent MHC-A localization in TSU-pr1 cells. Cells were grown on cover slips coated with collagen I, stimulated with EGF and samples taken at different time points, fixed, and prepared for indirect immunofluorescence using the MHC-A affinity-purified antibodies as described in Materials and Methods. In unstimulated TSU-pr1 cells (0 minutes), MHC-A was localized mainly in the cytoplasm. 1 minute after EGF stimulation, most of the MHC-A translocated to the cell cortex. The amount of MHC-A increased in the cortex with the time of EGF stimulation (2-10 minutes). Bar, 10 µm.

View larger version (81K): [in a new window]   Fig. 4. EGF-dependent MHC-B localization in TSU-pr1 cells. Cells were stimulated with EGF and prepared for indirect immunofluorescence as in Fig. 3. In unstimulated TSU-pr1 cells (0 minutes), MHC-B was localized mainly in the cytoplasm. After EGF stimulation, MHC-B gradually translocated to the cell cortex (1-2 minutes). 4 minutes after EGF stimulation, most of MHC-B localized at the cell cortex. 6 minutes after EGF stimulation, most of MHC-B localized in the cytoplasm. 10 minutes after EGF stimulation, there is another peak of MHC-B localization at the cell cortex. Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 5. PMA induced increases in MHC-A and MHC-B phosphorylation of TSU-pr1 cells, whereas calphostin C inhibited this phosphorylation. Images obtained from the PhosphorImager of MHC-A and MHC-B phosphorylation without any treatment (NT) and after the addition of 7 ng ml-1 EGF, 200 nM PMA or 7 ng ml-1 EGF and 100 nM calphostin C (calC). The experimental conditions and analysis of the phosphorylation levels were as in Fig. 3.

View larger version (47K): [in a new window]   Fig. 6. SDS-PAGE and western blot analysis of the expressed MHC-A and MHC-B tail domains. Purified MHC-A or MHC-B were prepared as described in Materials and Methods. Samples were subjected to SDS-PAGE on 12% gels and blotted on nitrocellulose. The immunoblots were stained with the MHC-A and MHC-B specific antibodies.

View larger version (29K): [in a new window]   Fig. 7. The effects of EGF, PMA and calphostin C on the activities of MHC-A and MHC-B kinase(s). Specific activity of MHC-A and MHC-B kinases, as determined by lysing TSU-pr1 cells treated with EGF, PMA or EGF and calphostin C, and subjecting them to MHCK assay as described in Materials and Methods. Error bars, ±s.e.m.; n=3-6.

View larger version (17K): [in a new window]   Fig. 8. Replacement of the MHC-A and MHC-B PKC phosphorylation sites with alanine residues prevented their phosphorylation by the EGF-activated MHC-A and MHC-B kinase(s). Specific activity of MHC-A and MHC-B kinases, as determined by lysing TSU-pr1 cells treated with EGF and subjecting them to MHCK assay using MHC-A, MHC-B, MHC-A S/A and MHC-B T/A tail domains as described in Materials and Methods. Error bars, ±s.e.m.; n=3.

View larger version (17K): [in a new window]   Fig. 9. Specific activity of MHC-A and MHC-B kinases(s) eluted from MHC-A and MHC-B tail domains affinity columns, respectively. Extracts from EGF-stimulated TSU-pr1 cells were loaded on MHC-A or MHC-B tail domain affinity columns. The columns were eluted in a stepwise manner with 100-250 mM NaCl, and the specific activity of MHC-A and MHC-B kinases was determined as described in Materials and Methods. (A-A), MHC-A kinase assayed with MHC-A tail fragment; (A-B), MHC-A kinase assayed with MHC-B tail fragment; (B-B), MHC-B kinase assayed with MHC-B tail fragment; (B-A), MHC-B kinase assayed with MHC-A tail fragment. Error bars, s.e.m.; n=6.