(Received for publication, November 15, 1995; and in revised form, January 18, 1996)
From the
Plectin is an in vitro substrate for various kinases
present in cell lysates from mitotic and interphase Chinese hamster
ovary cells. Sensitivity of plectin kinase activity to the inhibitor
olomoucine, and two-dimensional tryptic peptide mapping of plectin
phosphorylated by various kinase preparations suggested that the major
plectin kinase activity in mitotic extracts is related to the cell
cycle regulator kinase p34. Bacterial
expression of various truncated plectin mutant proteins comprising
different domains of the molecule and their phosphorylation by purified
p34
kinase revealed that the target site of
this kinase resided within plectin's C-terminal globular domain.
Among the subdomains of the C-terminal region (six repeats and a short
tail sequence), only repeat 6 and the tail were phosphorylated by
p34
kinase. As shown by two-dimensional
phosphopeptide mapping, repeat 6, but not the tail, contained a
mitosis-specific phosphorylation site targeted by p34
kinase in intact plectin molecules. By performing
site-directed mutagenesis of a potential p34
recognition sequence motif within the repeat 6 domain,
threonine 4542 was identified as the major target for the kinase.
Protein kinase A, phosphorylating plectin also within repeat 6,
targeted sites that were clearly different from those of
p34
kinase.
Plectin is an abundant cytoskeletal protein of exceptionally
large size. Electron microscopy of purified plectin molecules (1) and structure prediction based on the cloning and
sequencing of rat plectin cDNA (2) revealed an extended central
rod and two flanking globular domains as distinctive structural
features. Its subcellular distribution, in particular its partial
codistribution with intermediate filaments (IFs) ()and
prominent occurrence at plasma membrane attachment sites of IFs and
microfilaments, and the identification of numerous specific binding
proteins at the molecular level (reviewed in (3) and (4) ) suggested that plectin might be involved in versatile
cytoplasmic cross-linking functions. In a first approach to
characterize plectin's various binding domains, transient
transfection of mammalian cells using cDNAs encoding plectin mutant
proteins indicated a role of the C-terminal globular domain in the
binding to vimentin(5) .
As a prominent phosphoprotein
plectin was found to be an in vivo target of a
Ca/calmodulin-dependent kinase and of protein kinases
A and C(6, 7, 8) . In vitro studies
demonstrated that plectin's capacity to bind to IF proteins, such
as vimentin and lamin B, were differentially influenced by
phosphorylation(8) , suggesting that distinct protein kinases
were involved in regulating at least some of plectin's
interactions.
In view of plectin's proposed role as a
cytoplasmic cross-linking element, a specific regulation of its binding
activities would seem of particular importance during mitosis, when
dramatic structural rearrangements of the cytoskeleton, including IF
networks, take place. In fact, two of plectin's well
characterized binding partners, vimentin and lamin B, have been shown
to act as direct targets of mitotic cyclin-dependent p34 kinase. Phosphorylation of vimentin subunits by
p34
kinase at the onset of mitosis has been
shown to correlate with the disassembly of the vimentin
network(9, 10) , and the phosphorylation of lamin B by
p34
is directly related to the disassembly of
the nuclear lamina occurring at the same time, as demonstrated in
vivo(11, 12) and in
vitro(13, 14) . We report here that plectin, too,
serves as specific substrate of p34
kinase, and
we show that a single threonine residue residing in the C-terminal
globular domain serves as a target site.
Figure 3:
In vitro phosphorylation of
plectin mutant proteins by various kinases. Truncated plectin mutant
proteins encoded by the plasmids indicated were expressed in bacteria
using the pIMS (pWEI1, pWEI2, pWEI3) or pMAL-c (all others) expression
vector systems. Recombinant mutant proteins were subjected to in
vitro phosphorylation by various kinases as described in the text. cdc2, immunoprecipitated p34 kinase; PKA, protein kinase A; PKC, protein kinase C. +,
phosphorylation; -, no phosphorylation; nd, not
determined. Numbers in scheme on top mark positions of hydroxy
amino acid residues contained in potential recognition motifs for
p34
kinase. Asterisks denote
constructs modified by site-directed mutagenesis (see
text).
Figure 1:
In vitro phosphorylation of
plectin and histone H1. A, plectin, isolated from rat glioma
C cells (upper panel) and histone H1 (lower
panel) were phosphorylated in vitro using various kinase
preparations as indicated, and analyzed by SDS-PAGE and
autoradiography. Kinase preparations were cell lysates of
nocodazole-arrested mitotic cells (M-phase),
nocodazole-treated interphase cells (G2-phase), or cells in S
phase (S-phase), immunoprecipitates from these cell lysates
using antiserum to p34
(anti-cdc2) or
unspecific calf serum (mock), and precipitates from these cell
lysates using p13
-Sepharose beads. Control
lanes were incubated in the absence of plectin or histone H1. Coomassie
staining is shown in the first lanes; all others are autoradiographs. B, autoradiographs of plectin and histone H1 phosphorylated by
immunoprecipitated p34
kinase (cdc2),
p13 affinity-purified kinase (p13), protein kinase A (PKA), or protein kinase C (PKC), in the absence
(-) or presence (+) of the Cdc2-specific inhibitor
olomoucine.
To examine
whether plectin became phosphorylated at similar sites in vivo and in vitro, two-dimensional tryptic peptide mapping was
performed. Two of the spots generated from plectin immunoprecipitated
from metabolically labeled mitotic CHO cells (Fig. 2, panel
3, spots a and b) were also seen in plectin
phosphorylated in vitro by kinase activities contained in the
mitotic extract (Fig. 2, panel 4). This indicated that
some of the in vivo target sites of mitotic kinases were
recognized also in vitro. Peptide maps generated from purified
rat glioma C cell plectin phosphorylated with purified
kinases A (Fig. 2, panel 1) or C (Fig. 2, panel 2) showed different patterns, suggesting that these
kinases mainly affected plectin sites that were not phosphorylated by
mitotic kinases under in vivo conditions; furthermore, mitotic
cell lysates apparently did not contain any activities related to
kinases A and C. Purified C
cell plectin phosphorylated by
immunoprecipitated p34
kinase revealed two major
peptides, a and b (Fig. 2, panel 5),
both of which comigrated with the major spots generated from samples
phosphorylated by mitotic extracts (Fig. 2, panel 6).
This strongly suggested that plectin is a prominent target of
p34
kinase contained in mitotic cell lysates.
Furthermore, since these two major phosphopeptides were also present in
digests of mitotic samples labeled in vivo (Fig. 2, panel 3, spots a and b; and data not shown),
we concluded that purified p34
kinase phosphorylated
plectin in vitro at sites, which are similar to those targeted in vivo.
Figure 2:
Two-dimensional tryptic peptide maps of
plectin phosphorylated in vivo and in vitro by
different kinases. Tryptic peptide maps of metabolically labeled and
immunoprecipitated plectin (panel 3), or of plectin samples
phosphorylated in vitro by protein kinases A (panel
1) or C (panel 2), or by mitotic extract (panel
4), or by purified p34 kinase (panel 5)
are shown. In panel 6 equal amounts (cpm) of samples used for panels 4 and 5 were mixed and analyzed.
Phosphopeptides were separated by electrophoresis (bottom,
+; top, -) and chromatography (right to left), with the starting point in the lower right-hand
corner. Letters indicate the corresponding spots on
autoradiographs.
Figure 4:
In vitro phosphorylation of
mutant proteins corresponding to N-terminal or various C-terminal
repeat and tail domains. Mutant proteins encoded by pNM9 (lane
1), pTF15 (lane 2), pTH5 (lane 3), pNM1 (lane 4), pNM2 (lane 5), and pTH6 (lane 6)
were expressed in E. coli, using the pMAL-c expression system.
Cell lysates containing recombinant proteins, were subjected to
phosphorylation using immunoprecipitated p34 kinase, protein kinase C, or protein kinase A, as indicated.
Coomassie staining and autoradiography are shown. Arrowheads indicate expected sizes of fusion proteins; numbers, M
10
.
Figure 5:
Tryptic peptide maps (autoradiography) of
plectin and recombinant plectin mutant proteins phosphorylated by
p34 kinase. Panels 1 and 4, plectin
purified from rat glioma C
cells; panel 2,
pTH6-encoded mutant protein; panel 3, mixture of samples shown
in panels 1 and 2; panel 5, pNM10-encoded
mutant protein; panel 6, mixture of samples shown in panels 4 and 5. Phosphopeptides were separated by
electrophoresis (bottom, +; top, -) and
chromatography (right to left), with the starting
points in the lower right-hand corners. Letters indicate the corresponding spots on
autoradiographs.
Experiments using mutant proteins representing
truncated versions of repeat 6, containing either one of the two
p34 recognition motifs identified (Fig. 3, pNM4 and pNM5), showed that only the polypeptide
encoded by pNM4, containing the recognition motif TPGR, served as a
target for p34
kinase (Fig. 6, cdc2
kinase, lanes 2 and 4). Site-directed
mutagenesis of the threonine within the recognition motif to
isoleucine, led to a mutant protein that was no longer phosphorylated
upon incubation with p34
kinase (Fig. 6).
Figure 6:
p34 kinase phosphorylation
of plectin mutant proteins containing Ile instead of Thr at position
4542. Mutant proteins encoded by expression plasmids pNM1 (lane
1), pNM4 (lane 2), pNM5 (lane 4), and pNM6 (lane 3) were expressed in bacteria and phosphorylated by
p34
kinase or protein kinase A as indicated. Note that
only constructs containing Thr-4542 were phosphorylated by
p34
kinase. Coomassie staining and
autoradiography are shown. Arrowheads denote expected sizes of
fusion proteins; numbers, M
10
.
Figure 7:
Tryptic phosphopeptide maps of rat plectin
and plectin mutant proteins phosphorylated by various kinases. Panel 1, kinase A-phosphorylated rat plectin; panel
2, p34-phosphorylated rat plectin; panel
3, mixtures of samples shown in panels 1 and 2; panel 4, kinase A-phosphorylated repeat 6 encoded by the
plasmid pNM1; panel 5, kinase A-phosphorylated mutated repeat
6 encoded by plasmid pNM7; panel6, mixture of
samples shown in panels 1 and 4. Phosphopeptides were
separated by electrophoresis (bottom, +; top,
-) and chromatography (right to left), with the
starting point in the lower right-hand corner. Letters indicate the corresponding spots on
autoradiographs.
In this work we demonstrate that plectin is phosphorylated by
immunoprecipitated p34 kinase at a unique site in its
C-terminal domain. Although one cannot completely eliminate the
possibility that the purified immunoprecipitated p34
kinase used in this study contained minor contaminations of
coprecipitating kinases, for various reasons it is very likely that
p34
kinase activity is responsible for the
phosphorylation of plectin in our assays. 1) Immunoprecipitation of
p34
kinase was performed under stringent conditions
(0.1% SDS, 1% Triton X-100) using antibodies directed against a
C-terminal amino acid sequence unique for p34
;
mock-precipitated samples using unspecific antibodies did not exhibit
any kinase activity. 2) Immunoprecipitated p34
kinase
samples showed high H1 kinase activities and, unlike protein kinases A
and C, were efficiently inhibited by the specific inhibitor olomoucine (Fig. 1). 3) Immunoprecipitation from interphase cell extracts
did not yield histone H1 nor plectin kinase activities (Fig. 1),
being consistent with the presence of inactive p34
kinase during interphase. 4) Mutation (Thr
Ile) of a
potential p34
kinase phosphorylation site within the
repeat 6 domain of plectin diminished its phosphorylation by
p34
kinase, but not by protein kinase A ( Fig. 3and Fig. 6). 5) p34
kinase prepared
by affinity chromatography on p13
-Sepharose or by ion
exchange chromatography on DE-52 columns phosphorylated plectin at the
same sites as immunoprecipitated kinase ( Fig. 1and data not
shown).
Comparison of phosphopeptide maps generated from samples
phosphorylated in vitro using mitotic cell lysates versus purified p34 kinase suggested the major sites
phosphorylated to be the same in both cases. Thus, p34
kinase seems to represent the main activity among all plectin
kinase activities present in mitotic cell extracts. The phosphorylation
sites recognized by p34
kinase in vitro are
likely to represent genuine physiological targets, since the same sites
were phosphorylated in vivo. The majority of phosphorylation
sites affected by kinases C and A, on the other hand, were not detected
in samples phosphorylated in vivo, nor in plectin
phosphorylated by mitotic extracts, indicating that these kinases were
not activated during the normal growth and division cycle of CHO cells.
The phosphopeptide pattern of purified plectin after p34 kinase-treatment revealed two different spots (a and b), indicating two different phosphorylated sites. To map
these sites, we used the bacterial expression system pMAL-c, in which
the recombinant peptide is expressed fused to maltose-binding protein
(MBP). The relative large size of the MBP (
40 kDa) was shown to
have no effect on the ability of the recombinant proteins to serve as
substrates for various kinases, because proteins without MBP (after
cleavage with factor Xa) behaved in the same way. Of all the different
plectin domains tested, only the C-terminal part of the molecule,
containing repeat 6 and/or the 3` tail domain, proved to be
phosphorylated by p34
kinase. When tested without the
repeat 6 domain, the tail showed by far a stronger signal and became
the first candidate for closer investigations. Even though it did not
contain any of the reported p34
consensus
motifs(24) , it had numerous phosphate accepting residues (21
serines and 5 threonines). However, when the phosphopeptide pattern
derived from the tail was compared to that of the intact full-length
protein, it turned out that none of the phosphopeptides from one source
had a matching counterpart in the other. The reason why the tail, when
part of the whole molecule, was not phosphorylated, despite
constituting such a good in vitro substrate, probably was
limited accessibility in the native molecule. This assumption was
corroborated by the observation that in larger mutant proteins,
containing the tail and several of the preceding repeat domains,
tail-specific phosphorylation decreased and phosphopeptide patterns
resembled that of the full-length protein.
The finding that repeat
6, but not the tail domain, seemed to be the natural target of
p34 kinase was consistent with the fact that the only
perfect p34
consensus sequence motif found in the
C-terminal domain resided within repeat 6. Deletion and site-specific
mutagenesis confirmed this site as a phosphoacceptor of p34
kinase. The localization of a second phosphorylation site,
suggested by the appearance of peptide b in tryptic peptide
maps of intact plectin, is not clear, since none of the recombinant
plectin domains, which were able to serve as substrate for p34
kinase in vitro, revealed this spot in two-dimensional
phosphopeptide analysis. This discrepancy could be explained in two
ways. 1) There is in fact only one site and the digest of the total
plectin molecule may have been incomplete, so that the second spot
would represent a peptide phosphorylated at the same site but migrating
to a different position because of its larger size. 2) The
phosphorylation of the second site might be dependent on
post-translational modifications of the protein, which would not occur
in the bacterially expressed proteins, but could be relevant for
plectin purified from rat glioma C6 cells.
The situation that a
protein like plectin, containing an -helical double-stranded
coiled-coil rod domain flanked by globular domains, is preferentially
phosphorylated by p34
kinase in the presumably less
ordered domains adjacent to its rod applies also to the IF proteins
lamin (12, 25) and vimentin(10, 26) .
Since their phosphorylation by p34
kinase has been
implicated in the regulation of filament structure and assembly state,
it remains an intriguing question to what extent plectin's
structure and functions are influenced by p34
phosphorylation.