©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Plectin as a Substrate of p34Kinase and Mapping of a Single Phosphorylation Site (*)

(Received for publication, November 15, 1995; and in revised form, January 18, 1996)

Nicole Malecz Roland Foisner Christine Stadler Gerhard Wiche

From the Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 p34kinase 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.


INTRODUCTION

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 (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.


MATERIALS AND METHODS

Cell Culture, Synchronization and Metabolic Labeling

Chinese hamster ovary cells (clone CHO K1) were grown to a density of about 80% in plastic culture dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS (Life Technologies, Inc., Paisley, United Kingdom) and 50 µg/ml penicillin and streptomycin (Life Technologies, Inc.). For synchronization cells were arrested at G(1)/S by an overnight incubation in the presence of 2 mM thymidine (15) in Joklik's modified minimum essential medium (S-MEM, Life Technologies, Inc.) supplemented with 10% FCS, a nonessential amino acid mix (1:100, Life Technologies, Inc.), 25 mM Hepes/NaOH, pH 7.4, and 50 µg/ml penicillin and streptomycin. To obtain mitotic cells, they were released from the thymidine block for 3 h in complete S-MEM and allowed to grow for another 4 h in S-MEM containing 0.2 µg/ml nocodazole. For cells enriched in S phase, the cultures were released from the thymidine block in complete S-MEM for 4 h. Cells were washed with phosphate-free DMEM (Sigma, Deisenhofen, Germany) and metabolically labeled with 0.2 mCi/ml [P]orthophosphate (carrier-free, DuPont NEN, Dreieich, Germany) in phosphate-free DMEM supplemented with 25 mM Hepes/NaOH, pH 7.4, and 10% FCS dialyzed against 25 mM Hepes/NaOH, pH7.4 for 2 h. Nocodazole-arrested mitotic cells were harvested by mechanical agitation; cells still attached after nocodazole treatment (mainly in G(2) phase), as well as cells in S phase were scraped off. Interphase cell pellets from one 75-cm^2 Petri dish or mitotic cell pellets collected from three Petri dishes were washed in phosphate-buffered saline and dissolved in 300 µl of buffer A (50 mM Hepes/NaOH, pH 7.0, 5 mM MgCl(2), 1 mM EGTA, 0.5% Triton X-100, 100 mM NaCl, 0.1 mM dithiothreitol) supplemented with (i) protease inhibitors: PMSF (1 mM), benzamidine (10 mM), aprotinin (10 µg/ml), pepstatin (10 µg/ml), and leupeptin (10 µg/ml); (ii) phosphatase inhibitors: microystin (10 µM), okadaic acid (1 µM), calyculin (100 nM) (all from Life Technologies, Inc.), NaF (10 mM), sodium pyrophosphate (10 mM), beta-glycerophosphate (5 mM), and sodium orthovanadate (1 mM); and (iii) kinase inhibitors: 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7, 300 µM) and staurosporine (2 µM) (both from Sigma, Deisenhofen, Germany). Lysates were incubated with 500 µg/ml DNase and 200 µg/ml RNase (both from Boehringer, Mannheim, Germany) for 10 min, and 30 µl of 10 times RIPA buffer (1 times RIPA: 50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA) and 1% SDS were added. After addition of 1.5 ml of 1 times RIPA buffer samples were processed for immunoprecipitation. Part of the cell lysates were mixed with SDS electrophoresis sample buffer (16) and subjected to SDS-PAGE.

Immunoprecipitation

For immunoprecipitation 1 ml of each cell lysate in RIPA buffer was incubated with 10 µl 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 µl of antiserum to plectin (17) at 4 °C. 100 µl 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 times 10-s Ultra-turrax treatment at maximum speed. The lysates were incubated with 50 µg/ml DNase and 20 µg/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 µl 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 kinase, 1 ml of mitotic cell lysate was mixed with 100 µl of 10 times RIPA buffer plus 0.1% SDS, and incubated overnight at 4 °C with 20 µl of a rabbit antiserum generated against a synthetic peptide representing the C terminus of human p34 kinase (kindly provided by L. Gerace). The immunocomplex was precipitated by a 2-h incubation with 200 µl 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 5` primer carried an additional EcoRI site, and the 3` 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. 3are 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; (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.


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).



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 5` 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 beta-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 µg/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 (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 µg/ml DNase, and 20 µg/ml RNase using a glass/glass homogenizer. After addition of 600 mM NaCl, 8 mM beta-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 beta-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 µl of the plectin sample (0.1-0.2 mg/ml 10 mM sodium borate/NaOH, pH 8.9, 1 mM PMSF), or 10 µg histone H1 (Boehringer), or 30 µl of E. coli lysates (containing the mutant proteins) were mixed with 5 µl of total cell extracts, or 5 µl of immunoprecipitated or p13 affinity-purified p34 kinase fractions, and 5 µl of 10 times kinase buffer, supplemented with 10 µM microcystin and protease inhibitor mixture. The phosphorylation reaction, started by adding 50 µM ATP and 5 µCi of [-P]ATP, was carried out for 30-90 min at 30 °C, and stopped by adding 3 times SDS-PAGE sample buffer(16) . Phosphorylation with protein kinase A and protein kinase C was done as described elsewhere(8) . To inhibit p34 kinase activity, 0.5 mM olomoucine (Promega Corp., Madison, WI; (22) ) were added to the incubation mixture.

Two-dimensional Tryptic Phosphopeptide Mapping

Phosphorylated samples were separated by SDS-PAGE and processed according to (23) , except that gel pieces containing the protein (prepared as described in (14) ) were used for trypsin digestion.


RESULTS

Plectin Is a Substrate for Mitotic p34-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 > 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 kinase, was one of the major kinase activities present in this lysate. To investigate whether p34 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 (1 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 (Fig. 1A, p13). These experiments suggested that plectin can serve as a direct in vitro substrate for mitotic p34 kinase. In contrast to immunoprecipitated samples, such activities were contained also in p13 purified kinase preparation from S phase cells, indicating that non-mitotic Cdk kinases distinct from p34 might also phosphorylate these proteins (Fig. 1A, p13). The plectin and histone H1 kinase activities of immunoprecipitated (Fig. 1B, cdc2) as well as p13-precipitated kinases (Fig. 1B, p13) 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 kinase.


Figure 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 (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(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 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.



Localization of the p34 Phosphorylation Sites

The consensus p34 recognition motif (S/T)-P-X-(K/R) (24) can be found twice in plectin's polypeptide chain ( (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-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 kinase ( Fig. 3and 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 ( Fig. 4and data not shown). To address the question why the tail domain served as a good substrate for p34 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 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 two-dimensional peptide mapping, one of the spots appeared to 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 kinase (Fig. 4) and the tail showed a different pattern compared to intact plectin, the p34 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.


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(r) times 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(6) 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(r) times 10.



The p34 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; Fig. 3and Fig. 4, lane 3). Since kinase A and p34 kinase both phosphorylated plectin within the repeat 6 domain, it was of interest to test whether they targeted the same sites. A comparison of two-dimensional peptide maps generated from total plectin phosphorylated by p34 (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-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-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).


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.




DISCUSSION

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 alpha-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.


FOOTNOTES

*
This study was supported by grants from the Austrian Science Research Fund (to R. F. and G. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: IF, intermediate filament; MBP, maltose-binding protein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; S-MEM, Joklik's modified minimum essential medium; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; RIPA, radioimmune precipitation buffer; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Larry Gerace (Scripps Clinic Research Institute, La Jolla, CA) for donating antiserum to p34 kinase and Heribert Hirt (University of Vienna), for providing a bacterial strain overproducing p13.


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