Identification of plectin as a substrate of p34cdc2 kinase and mapping of a single phosphorylation site.

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 p34cdc2. Bacterial expression of various truncated plectin mutant proteins comprising different domains of the molecule and their phosphorylation by purified p34cdc2kinase 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 p34cdc2 kinase. As shown by two-dimensional phosphopeptide mapping, repeat 6, but not the tail, contained a mitosis-specific phosphorylation site targeted by p34cdc2 kinase in intact plectin molecules. By performing site-directed mutagenesis of a potential p34cdc2 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 p34cdc2 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) 1 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 Refs. 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 2ϩ /calmodulin-dependent kinase and of protein kinases A and C (6 -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 crosslinking 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 cdc2 kinase. Phosphorylation of vimentin subunits by p34 cdc2 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 cdc2 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 cdc2 kinase, and we show that a single threonine residue residing in the C-terminal globular domain serves as a target site.
Immunoprecipitation-For immunoprecipitation 1 ml of each cell lysate in RIPA buffer was incubated with 10 l of protein A-Sepharose (Pharmacia Biotech, Inc., Brussels, Belgium; 10% (w/v) in RIPA buffer) for 1 h, centrifuged for 1-2 min in an Eppendorf centrifuge, and supernatants incubated overnight with 10 l of antiserum to plectin (17) at 4°C. 100 l of 10% protein A-Sepharose were added to each sample and incubated for another 4 h. After a 5-min centrifugation, the pellets were washed three times in RIPA buffer plus 0.1% SDS and dissolved in electrophoresis sample buffer.
Preparation of Protein Kinases-Nocodazole-arrested CHO cells (grown in four 900-cm 2 roller bottles) or S phase cells (grown in one roller bottle) were collected and lysed in 1 ml of buffer A (containing protease inhibitor mixture) by 3 ϫ 10-s Ultra-turrax treatment at maximum speed. The lysates were incubated with 50 g/ml DNase and 20 g/ml RNase for 10 min and centrifuged for 20 min at 35,000 rpm in a Beckman 65 rotor (Beckman Instruments Inc. Palo Alto, CA). Mitotic and interphase cell lysates were diluted to the same protein concentration, mixed with 16% glycerol, and frozen in liquid nitrogen. Cdk type kinases were isolated from the cell lysate supernatants by incubation with 100 l of p13-Sepharose beads (18) overnight at 4°C. The beads were sedimented, washed in kinase buffer (20 mM Hepes/NaOH, pH 7.0, 10 mM MgCl 2 ) and frozen in the same buffer, containing 16% glycerol. For the preparation of the p34 cdc2 kinase, 1 ml of mitotic cell lysate was mixed with 100 l of 10 ϫ RIPA buffer plus 0.1% SDS, and incubated overnight at 4°C with 20 l of a rabbit antiserum generated against a synthetic peptide representing the C terminus of human p34 cdc2 kinase (kindly provided by L. Gerace). The immunocomplex was precipitated by a 2-h incubation with 200 l of 10% protein A-Sepharose, washed in kinase buffer, and frozen in this buffer plus 16% glycerol.
cDNA Constructs-To obtain cDNA inserts for the plasmids pNM1, pNM2, pNM4, pNM5, pTH4, pTF15, and pTH6, PCR under standard conditions (19) was performed using plasmid pAD14 (5) as template. Primers were constructed in such a way that the 5Ј primer carried an additional EcoRI site, and the 3Ј primer an additional XbaI site, to facilitate site-directed cloning. PCR products were cloned into the pMAL-c expression vector (New England Biolabs, Beverley, MA). The plectin coordinates given in Fig. 3 are based on the numbering according to a revised rat plectin sequence (data not shown), in which a new translation start codon has been identified (1635 bp upstream of the originally published ATG; Ref. 2). pTH1 was constructed by cloning a cDNA insert, covering the region from the new ATG to bp 3384, into pMAL-c; for pNM9 a cDNA insert, covering the N-terminal region from bp 1636 to 3384, was used. To obtain pNM10, a 5800-bp NdeI/XhoI fragment of pTH4, containing 2550 bp encoding repeats 4 and 5 and part of repeat 6, was ligated to a 3047-bp NdeI/XhoI fragment of pNM2 encoding 483 bp of the end of repeat 6 and the tail domain.
Site-directed Mutagenesis-To change the threonine in position 4542 to an isoleucine, the method of gene splicing by overlap extension (20) was adopted using pAD14 as template and internal primers that contained an ATA instead of an ACA codon. The final PCR product corresponding to part of repeat 6 cDNA (bp 4368 -4597) was cloned into pMAL-c, giving rise to pNM6. To obtain pNM7, encoding mutagenized repeat 6 as a whole, a 2654-bp SacII/NdeI fragment of pNM 1 (62 bp of which encode the 5Ј end of repeat 6) was ligated to a 4381-bp fragment of pNM6 (containing 825 bp of mutagenized repeat 6 cDNA).
Expression of cDNA-encoded Proteins-Two bacterial expression systems were used, pMAL-c (New England Biolabs) and pIMS (21). pMAL-c vector constructs were expressed in Escherichia coli strain HB 101. Expression was induced in cultures grown to the end of the logarithmic phase for 2 h with 0.1 mM isopropyl ␤-D-thiogalactopyranoside. Cells were harvested and lysed in 20 mM Tris/HCl, pH 7.4, 0.2 M NaCl, 1 mM EGTA, 0.2% Tween 20, 10 mM benzamidine, and 10 g/ml aprotinin, leupeptin, and pepstatin, by sonication. Overexpressed fusion proteins were purified on amylose columns (New England Biolabs) as described by the manufacturer. pIMS cDNA constructs encoding mutant proteins corresponding to clones C1 and C2 combined, C5 and C3 (described in Ref. 2) were expressed in E. coli strain XL-1 blue (Stratagene Inc., La Jolla, CA) as described (2). Cell pellets, frozen in liquid nitrogen, were ground in a mortar in the presence of 30 mg of solid lysozyme (Sigma) per gram of wet cells and homogenized for 10 min in 10 volumes of phosphate-buffered saline plus 100 mM MgCl 2 , 50 g/ml DNase, and 20 g/ml RNase using a glass/glass homogenizer. After addition of 600 mM NaCl, 8 mM ␤-mercaptoethanol, and 1% Triton X-100, inclusion bodies with the overexpressed proteins were collected by centrifugation for 20 min at 19,000 rpm in a Sorvall SS 34 rotor and solubilized in 10 volumes (w/v) of 50 mM sodium borate, pH 8.7, 0.1% sodium lauryl sulfate, 5 mM EDTA, 4 mM ␤-mercaptoethanol, 1 mM PMSF. Solubilized proteins were subjected to Sephacryl S-300 gel permeation column chromatography (Pharmacia, Uppsala, Sweden).
In Vitro Phosphorylation-Plectin was purified from rat glioma C6 cells as described elsewhere (1). 30 l of the plectin sample (0.1-0.2 mg/ml 10 mM sodium borate/NaOH, pH 8.9, 1 mM PMSF), or 10 g histone H1 (Boehringer), or 30 l of E. coli lysates (containing the mutant proteins) were mixed with 5 l of total cell extracts, or 5 l of immunoprecipitated or p13 affinity-purified p34 cdc2 kinase fractions, and 5 l of 10 ϫ kinase buffer, supplemented with 10 M microcystin and protease inhibitor mixture. The phosphorylation reaction, started by adding 50 M ATP and 5 Ci of [␥-32 P]ATP, was carried out for 30 -90 min at 30°C, and stopped by adding 3 ϫ SDS-PAGE sample buffer (16). Phosphorylation with protein kinase A and protein kinase C was done as described elsewhere (8). To inhibit p34 cdc2 kinase activity, 0.5 mM olomoucine (Promega Corp., Madison, WI; Ref. 22) were added to the incubation mixture.
Two-dimensional Tryptic Phosphopeptide Mapping-Phosphorylated samples were separated by SDS-PAGE and processed according to Ref. 23, except that gel pieces containing the protein (prepared as described in Ref. 14) were used for trypsin digestion.

RESULTS
Plectin Is a Substrate for Mitotic p34 cdc2 -Kinase-To analyze plectin kinase activities at different stages of the cell cycle, we prepared total cell lysates from CHO cell cultures enriched in mitotic cells (mitotic index Ͼ 90%), or from cultures predominantly containing cells in S or in G 2 phase. Kinase activities were analyzed using purified plectin and histone H1 as in vitro substrates (Fig. 1A, Cell Lysates). Unlike histone H1 (Fig. 1A, lower panels), plectin was phosphorylated by kinases contained in all three cell lysates to a similar extent (Fig. 1A, upper panels), suggesting that plectin served as substrate for these protein kinases throughout the cell cycle. Control experiments performed in the absence of exogenous plectin (Fig. 1, Control) showed that endogenous plectin was not detectable in autoradiographs. The high level of histone H1 kinase activity in mitotic cell lysates further suggested that the mitotically active Cdk, p34 cdc2 kinase, was one of the major kinase activities present in this lysate. To investigate whether p34 cdc2 kinase was able to phosphorylate plectin directly, the kinase was immunoprecipitated from mitotic and interphase cell lysates and its activity tested using plectin and histone H1 as substrates. The kinase immunoprecipitated from mitotic cell lysates showed a high histone H1 kinase activity, and, unlike kinases immunoprecipitated from S phase cell lysates and mock-precipitated samples, it phosphorylated plectin to a relatively high extent (ϳ1 mol of phosphate/mol of plectin) (Fig.  1A, Immunoprecipitates; and data not shown). Samples immunoprecipitated from G 2 phase lysates also showed histone H1 and plectin kinase activities, probably due to remnants of mitotic cells in the preparation. Significant phosphorylation of both plectin and histone H1 was observed also with protein kinase preparations obtained from mitotic cell lysates by affinity purification on immobilized p13 suc1 (Fig. 1A, p13 suc1 ). These experiments suggested that plectin can serve as a direct in vitro substrate for mitotic p34 cdc2 kinase. In contrast to immunoprecipitated samples, such activities were contained also in p13 suc1 purified kinase preparation from S phase cells, indicating that non-mitotic Cdk kinases distinct from p34 cdc2 might also phosphorylate these proteins (Fig. 1A, p13 suc1 ). The plectin and histone H1 kinase activities of immunoprecipitated (Fig.  1B, cdc2) as well as p13 suc1 -precipitated kinases (Fig. 1B, p13) p34 cdc2 Phosphorylation of Plectin were significantly reduced in the presence of olomoucine, an inhibitor specific for Cdk-type kinases. Activities of protein kinase A and protein kinase C were much less affected by this inhibitor (Fig. 1B, PKA and PKC), confirming that the isolated mitotic plectin kinase activity represented genuine p34 cdc2 kinase.
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 6 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 6 cell plectin phosphorylated by immunoprecipitated p34 cdc2 ki-nase 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 cdc2 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 cdc2 kinase phosphorylated plectin in vitro at sites, which are similar to those targeted in vivo.
Localization of the p34 cdc2 Phosphorylation Sites-The consensus p34 cdc2 recognition motif (S/T)-P-X-(K/R) (24) can be found twice in plectin's polypeptide chain (Ref. 2 and data not shown). One of the sites (SPAK) is located in the rod-domain, the other (TPGR) in repeat 6 of the C-terminal globular domain; in addition, repeat 6 contains a slightly degenerate motif (SPYS) (Fig. 3). To map the p34 cdc2 -specific phosphorylation site(s), recombinant plectin mutant proteins, corresponding to different domains of the molecule (Fig. 3), were expressed in bacteria and used as in vitro substrates for the kinase. It was found that only those mutant peptides that contained repeat 6 and/or the C-terminal tail domain were phosphorylated by p34 cdc2 kinase (Figs. 3 and 4). Mutant peptides containing the N-terminal region, plectin's rod domain, or the first three repeats of the C-terminal domain were not recognized by p34 cdc2 (Fig. 4 and data not shown). To address the question why the tail domain served as a good substrate for p34 cdc2 kinase, even though it did not contain a consensus recognition sequence motif, two-dimensional peptide mapping was performed. Spots a and b, seen in the phosphopeptide pattern of the intact molecule after p34 cdc2 phosphorylation (Fig. 5, panel 1), were missing in the peptide map derived from the mutant protein containing just the tail domain (pTH6). However, in the latter case numerous additional peptides appeared instead, which were not part of the pattern observed with the whole molecule (Fig. 5, panels 2 and 3). This suggested that the phosphorylation sites in the tail region were not accessible to the kinase in the intact molecule and therefore did not constitute native target sites. When the mutant protein encoded by pNM10 (containing repeats 4 -6 and the tail) was subjected to twodimensional peptide mapping, one of the spots appeared to FIG. 1. In vitro phosphorylation of plectin and histone H1. A, plectin, isolated from rat glioma C 6 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 cdc2 (anti-cdc2) or unspecific calf serum (mock), and precipitates from these cell lysates using p13 suc1 -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 cdc2 kinase (cdc2), p13 affinity-purified kinase (p13), protein kinase A (PKA), or protein kinase C (PKC), in the absence (Ϫ) or presence (ϩ) of the Cdc2-specific inhibitor olomoucine. p34 cdc2 Phosphorylation of Plectin comigrate with peptide a, seen in intact plectin, while several other spots, not seen with the whole protein (Fig. 5, panels 4  and 6), seemed to be derived from the tail domain (Fig. 5, panel  5). Since repeats 4 and 5 were not phosphorylated by p34 cdc2 kinase (Fig. 4) and the tail showed a different pattern compared to intact plectin, the p34 cdc2 site corresponding to spot a was likely to represent a site within repeat 6. Peptide b, derived from intact plectin, however, was not detected in the tryptic peptide maps of any of the bacterially expressed mutant proteins, which served as substrates for p34 cdc2 .
Experiments using mutant proteins representing truncated versions of repeat 6, containing either one of the two p34 cdc2 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 cdc2 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 cdc2 kinase (Fig. 6).
The p34 cdc2 Site in Repeat 6 Is Not Recognized by Protein Kinases A and C-Protein kinase A phosphorylated mutant proteins encoded by pWEI2, pWEI3, pNM1, pNM2, and pTH6 at target sites residing in repeat 3, repeat 6, and the tail (Fig.  3), whereas protein kinase C recognized only a mutant protein corresponding to repeat 5 (encoded by pTH5; Figs. 3 and 4, lane 3). Since kinase A and p34 cdc2 kinase both phosphorylated plectin within the repeat 6 domain, it was of interest to test whether they targeted the same sites. A comparison of twodimensional peptide maps generated from total plectin phosphorylated by p34 cdc2 (Fig. 7, panel 2) or protein kinase A (Fig.  7, panel 1), revealed that none of the kinase A-specific spots, were comigrating with the p34 cdc2 -specific peptides a and b (Fig. 7, panel 3). Repeat 6 phosphorylated by protein kinase A gave rise to three spots (Fig. 7, panel 4, spots f-h), which were also seen in total plectin after phosphorylation with kinase A, but were clearly different from the p34 cdc2 -specific spot a. In support of this, the pNM6-encoded mutant protein, containing an isoleucine instead of a threonine at the TPGR sequence motif of repeat 6, continued to be a good substrate for protein kinase A (Fig. 6); furthermore, the two-dimensional peptide map of this mutated protein was indistinguishable from that of unmutated repeat 6, encoded by pNM1 (Fig. 7, panels 4 and 5). DISCUSSION In this work we demonstrate that plectin is phosphorylated by immunoprecipitated p34 cdc2 kinase at a unique site in its C-terminal domain. Although one cannot completely eliminate the possibility that the purified immunoprecipitated p34 cdc2 kinase used in this study contained minor contaminations of coprecipitating kinases, for various reasons it is very likely that p34 cdc2 kinase activity is responsible for the phosphorylation of p34 cdc2 Phosphorylation of Plectin plectin in our assays. 1) Immunoprecipitation of p34 cdc2 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 cdc2 ; mock-precipitated samples using unspecific antibodies did not exhibit any kinase activity. 2) Immunoprecipitated p34 cdc2 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 cdc2 kinase during interphase. 4) Mutation (Thr 3 Ile) of a potential p34 cdc2 kinase phosphorylation site within the repeat 6 domain of plectin diminished its phosphorylation by p34 cdc2 kinase, but not by protein kinase A (Figs. 3 and 6). 5) p34 cdc2 kinase prepared by affinity chromatography on p13 suc1 -Sepharose or by ion exchange chromatography on DE-52 columns phosphorylated plectin at the same sites as immunoprecipitated kinase ( Fig. 1 and data not shown).
Comparison of phosphopeptide maps generated from samples phosphorylated in vitro using mitotic cell lysates versus purified p34 cdc2 kinase suggested the major sites phosphorylated to be the same in both cases. Thus, p34 cdc2 kinase seems to represent the main activity among all plectin kinase activities present in mitotic cell extracts. The phosphorylation sites recognized by p34 cdc2 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 cdc2 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 (ϳ40 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 3Ј tail domain, proved to be phosphorylated by p34 cdc2 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 cdc2 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 fulllength 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 cdc2 kinase was consistent with the fact that the only perfect p34 cdc2 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 cdc2 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 cdc2 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 p34 cdc2 Phosphorylation of Plectin 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 cdc2 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 cdc2 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 cdc2 phosphorylation.