QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 3. Phosphopeptide mapping of the major interphase-specific in vivo phosphorylation sites on vimentin. (A) The relatively low level of ³²P-labeling of vimentin in interphase cells shown in Fig. 1, reflects constitutive phosphorylation on a few major sites, as indicated by the presence of a few more prominently labeled tryptic peptides (1, 4, 5, and 8) from vimentin isolated from untreated ³²P in vivo labeled BHK-21 cells. (B) When dephosphorylation is inhibited with 20 nM cl-A for 20 minutes, all (except peptide 8) of these constitutively labeled phosphopeptides displayed significant increases in labeling and, in addition, many new peptides (2, 3, 6, 7) showed marked elevations in ³²P labeling. These results indicate that there are some sites that maintain a certain level of constitutive phosphorylation and, in addition, some sites that are subjected to a high constitutive phosphate turnover. (C) The interphase-specific sites do not correspond to the mitosis-specific in vivo phosphorylation sites, as shown on a vimentin phosphopeptide map derived from in vivo labeled cells, blocked in metaphase by treatment with 2 µg/ml nocodazole for 3 hours. (D-H) The in vivo phosphopeptide maps were compared to those obtained by in vitro phosphorylation using a number of potential vimentin kinases. (D) PKA, (E) PKC, (F) CaMKII, (G) p37 K, and (H) cdc2 K, all resulted in characteristic phosphopeptide maps. Some of the major interphase-specific in vivo phosphopeptides showed a similar migration as the major phosphopeptides generated by PKA and PKC but not CaMK. The phosphopeptides generated by the mitotic kinases cdc2 K and p37 K did not correspond to any of the interphase-specific phosphopeptides but co-migrated with the major mitosis-specific phosphopeptides.

Return to article