Phosphorylation-induced disassembly of vimentin IFs in vitro and in vivo results in disassembled subunits with the same molecular mass categories. (A) Bacterially expressed human vimentin IF polymers were phosphorylated in vitro by PKA and the resultant disassembly of depolymerized subunits was assayed by glutaraldehyde crosslinking (GA; concentration range: 0-0.06%) of the vimentin supernatant fractions after 30 minutes centrifugation at 200,000 g. (B) The in vitro disassembly of vimentin was compared to that in vivo, by incubating BHK-21 cells in the presence of 50 nM cl-A for 30 minutes. The cells were treated with 1% Triton X-100 and the particulate material was pelleted by centrifugation at 200,000 g for 30 minutes. The supernatants were then subjected to glutaraldehyde crosslinking (same concentration range as above) followed by immunoblottting. The results indicate that the phosphorylation-mediated release of vimentin subunits also occurs as tetramers in vivo.

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.

Chromatographic separation of the major tryptic phosphopeptides from ³²P in vivo labeled vimentin. Four 20 cm plates of semi-confluent BHK-21 cells were preincubated for 4 hours with 0.5 mCi/ml [³²P]orthophosphate. In order to maximize the in vivo labeling, vimentin dephosphorylation was inhibited by addition of 50 nM cl-A to the cells. Vimentin was isolated by preparative SDS-PAGE as described in Materials and Methods and then subjected to tryptic cleavage. (A) The generated phosphopeptides were first isolated by reversed phase chromatography on a microbore C-18 column. The UV chromatogram (left panel) indicates the presence of a high number of tryptic peptides, among which seven ³²P labeled fractions were isolated (right panel; fractions a-g). (B) Peaks a-g obtained on the C18 chromatography were then separated a second time on a reversed phase microbore C-8 column, yielding one single purified peptide from fractions a, b and d-g, and two peptides from fraction c. The inserts show the elution profile of the ³²P label, which in all cases corresponded to a peak on the UV chromatogram. The isolated peptides were numbered according to their hydrophobicity on the C8 reversed phase HPLC. The obtained peaks were subjected to automatic sequencing and manual Edman degradation. Peptide masses were confirmed by mass-spectrometry (data not shown). The results are presented in Table 1.

Identified in vivo phosphorylation sites on vimentin. (A) A summary of all results obtained by phosphopeptide mapping, sequencing and manual Edman degradation. P, identified phosphorylation sites; *, sites that have not been previously described as in vivo phosphorylation sites. (B) The schematic figure shows the relative location of the phosphorylation sites in the N and C termini of vimentin.

Mutation of the two major PKA sites results in a reduction of vimentin phosphorylation and alters the phosphate distribution on vimentin phosphopeptides. When two-dimensional tryptic phosphopeptide mapping of PKA-phosphorylated (A) wild-type and (B) S38:A/S72:A vimentin was performed, peptide 1 showed negligible phosphorylation in the mutant vimentin, whereas peptide 2 showed reduced phosphorylation. Each map was loaded with peptides generated from 5 μg of ³²P-labeled vimentin. The direction of electrophoresis (+,-) and ascending chromatography (arrow) are indicated. (C) An overall reduction in ³²P incorporation as a result of the phosphate-site mutations could be seen when 7.5 μg of wild-type (lane 1) and S38:A/S72:A (lane 2) vimentin, respectively, were phosphorylated in vitro by PKA and subjected to one-dimensional SDS-PAGE followed by autoradiography.