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J. Biol. Chem., Vol. 277, Issue 4, 3053-3059, January 25, 2002

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Phosphorylation of the Myosin-binding Subunit of Myosin Phosphatase by Raf-1 and Inhibition of Phosphatase Activity^*

Constantinos G. Broustas, Nicholas Grammatikakis^§, Masumi Eto^¶, Paul Dent, David L. Brautigan^¶, and Usha Kasid^**

From the  Departments of Radiation Medicine and Biochemistry and Molecular Biology, Lombardi Cancer Center, Georgetown University, Washington, D. C. 20007, the ^§ Department of Physiology, Tufts University, Boston, Massachusetts 02111, the ^¶ Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908, and the  Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

Received for publication, July 6, 2001, and in revised form, November 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Raf-1 serine/threonine protein kinase plays an important role in cell survival, proliferation, and migration; however, the specific targets of Raf-1 in diverse cellular processes are not clearly defined. Myosin phosphatase activity is critical to the regulation of cytoskeletal reorganization, cytokinesis, and cell motility. Here, we describe the association of Raf-1 with myosin phosphatase and phosphorylation of the regulatory myosin-binding subunit (MBS) of myosin phosphatase by Raf-1. Treatment of cells with phorbol 12-myristate 13-acetate has been shown to stimulate Raf-1 protein kinase. To determine the effect of enzymatic activation of Raf-1 on MBS phosphorylation, COS-1 cells were transiently transfected with FLAG-tagged full-length Raf-1. A significantly higher phosphorylation of purified glutathione S-transferase-tagged truncated MBS protein (amino acids 654-880) occurred in the presence of FLAG-Raf-1 immunoprecipitated from phorbol 12-myristate 13-acetate-treated cells compared with untreated cells (~3.0-fold). Using a sequential kinase-phosphatase assay and phosphorylated myosin light chain as substrate in the phosphatase reaction, we showed that Raf-1-associated protein phosphatase-specific activity was inhibited (relative phosphatase activity without and with adenosine 5'-O-(3-thiotriphosphate): 100 and ~30%, respectively). Previously, ionizing radiation has been shown to activate Raf-1 (Kasid, U., Suy, S., Dent, P., Ray, S., Whiteside, T. L., and Sturgill, T. W. (1996) Nature 382, 813-816). Exposure of cells to ionizing radiation resulted in the increased association of Raf-1 with MBS (3-6-fold versus unirradiated control) and inhibition of Raf-1-associated protein phosphatase-specific activity (relative phosphatase activity without and with ionizing radiation: 100 and ~54%, respectively). Our studies identify MBS as a new substrate of Raf-1 and implicate a role for Raf-1 in the regulation of pathways involving myosin phosphatase activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Raf-1 serine/threonine protein kinase plays an important role in cell survival, proliferation, and migration (1-6); however, the specific targets of Raf-1 in diverse cellular processes are not clearly defined. Although MEK1 is the most widely recognized physiological substrate for Raf-1, growing evidence suggests a link between Raf-1 and a variety of other downstream effectors (7). Recently, Raf-1 has been linked with cytoskeletal architecture via its association with vimentin and vimentin kinases (8). These data underscore the importance of as yet unknown effectors likely to be involved in the Raf-1-mediated biological response.

Myosin phosphatase holoenzyme is a heterotrimer consisting of an ~130-kDa regulatory myosin-binding subunit (MBS)¹; an ~38-kDa catalytic protein phosphatase subunit, PP1c (PP1); and a 20-kDa protein of as yet unknown function, M20 (9, 10). MBS binds to both PP1 and phosphorylated myosin light chain (MLC^P), targeting PP1 to its substrate MLC^P and resulting in MLC dephosphorylation. MLC phosphorylation is essential for the contractile force of cell motility (11, 12). Myosin phosphatase activity is regulated through phosphorylation of MBS, and several kinases have been shown to modulate phosphatase activity via phosphorylation of MBS (13-16).

In efforts to identify novel Raf-1-interacting proteins, we discovered that Raf-1 associates with MBS under a variety of in vitro and in vivo experimental conditions, including radiation treatment. Raf-1 was found to phosphorylate MBS, and this association led to a concomitant inhibition of Raf-1-interacting protein phosphatase activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Radiation Treatment-- MDA-MB 231 human breast cancer cells (obtained from the Tissue Culture Resource of the Lombardi Cancer Center) were maintained and serum-starved overnight prior to irradiation as described (17, 18). COS-1 cells (obtained from the Tissue Culture Resource) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM glutamine, and 50 µg/ml gentamycin (all from Invitrogen) at 37 °C in an atmosphere of 95% air and 5% CO₂.

Plasmid Expression Vectors-- Plasmid DNA constructs for expression of GST fusion proteins of wild-type/full-length Raf-1 (GST-Raf-1) and its NH₂-terminal domain (amino acids 1-323, GST-Raf-N) were generated, and GST-Raf-1 fusion protein was purified as described (19). The constructs for expression of FLAG-tagged wild-type (FLAG-Raf-1) and mutant (FLAG-Raf-1(K375M)) Raf-1 in COS-1 cells were generated as described (20). A human MBS cDNA clone was isolated from a HeLa cell cDNA library using reverse transcription-PCR and inserted into a mammalian expression vector (pRK7) downstream of a Myc tag sequence to express NH₂-terminal Myc-tagged full-length MBS protein (Myc-MBS, ~140 kDa). A cDNA fragment encoding a portion of the human MBS protein (MBS, amino acids 654-880) was amplified using a pair of mismatch primers to introduce the flanking BamHI and EcoRI sites. The partial cDNA fragment was subcloned into the pGEX4T-2 vector (Amersham Biosciences, Inc.) for expression of GST-MBS fusion protein (~55 kDa). The sequences of full-length MBS cDNA and its fragment were confirmed by the dideoxy sequencing method at the Biomolecular Core Facility of the University of Virginia. GST-MBS fusion protein was expressed in Escherichia coli strain BL21(DE3) by incubation at 23 °C for 24 h using LB medium in the presence of 0.1 mM isopropyl-1-thio--D-galactopyranoside and purified by glutathione-agarose column chromatography according to the manufacturer's protocol (Amersham Biosciences, Inc.).

MBS Identification Procedure-- MDA-MB 231 cells (2.5 × 10⁸) were lysed in Nonidet P-40 lysis buffer (100 mM HEPES (pH 7.4), 10% glycerol, 150 mM NaCl, 1% Nonidet P-40, 50 mM NaF, 5 mM Na₃VO₄, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) as described (17). The supernatant was precleared for 1-2 h with protein A-Sepharose CL-4B (Amersham Biosciences, Inc.) and incubated with monoclonal anti-Raf-1 antibody for 6 h at 4 °C. Rabbit anti-mouse IgG was then added, and the immune complex was captured by overnight incubation with protein A-Sepharose CL-4B at 4 °C. The beads were washed with Nonidet P-40 lysis buffer, and samples were pooled and electrophoresed on 7.5% SDS-polyacrylamide gel. Following electrophoresis, the gel was stained without fixation with Coomassie Brilliant Blue R-250. The Coomassie Blue-stained bands (~130 and ~135 kDa) in various lanes were excised and electroeluted separately in an Amicon Centrilutor microelectroeluter (Millipore) in 25 mM Tris and 192 mM glycine (pH 8.3). The eluates were concentrated in a Centricon-30 concentrator (Millipore), pooled, and subjected to 6.0% SDS-PAGE. The proteins were detected by Coomassie Blue staining as described above; the gel was partially destained; and two bands representing a doublet (~130 and ~135 kDa) were excised separately, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry on an LCQ ion trap mass spectrometer (Finnigan MAT) at the Yale University Microsequencing Facility. Mass spectra of peptides from p130 and p135 were compared with the protein and gene sequence data bases using the SEQUEST computer program (21, 22).

Immunoprecipitation Assay-- MDA-MB 231 cells were lysed in Nonidet P-40 lysis buffer for 30 min in a cold room and centrifuged at 14,000 × g for 20 min. Two mg of protein was precleared over protein A/G Plus-agarose (Santa Cruz Biotechnology) for 1-2 h; the beads were removed by centrifugation; and the supernatant was incubated with a desired antibody overnight at 4 °C. The antigen-antibody complex was captured by incubation with 25 µl of protein A/G Plus-agarose for 1.5-3 h. The beads were washed three times with ice-cold Nonidet P-40 lysis buffer and once with 50 mM Tris-HCl (pH 7.4). The immune complex was used in an immunoblot and/or in vitro kinase assay.

Transient Transfections-- COS-1 cells were transiently transfected using LipofectAMINE reagent according to the manufacturer's protocol (Invitrogen). For each 100-mm culture dish, 10-15 µg of plasmid DNA was used. Forty-eight h after transfection, the cells were washed twice with ice-cold PBS and lysed in 0.5 ml of Nonidet P-40 lysis buffer (with 200 mM NaCl), and lysates were tested for expression of exogenous protein by immunoprecipitation and immunoblot assays.

GST Pull-down Assay-- COS-1 cells were transiently transfected with the expression vector containing Myc-MBS or the Myc tag control vector, followed by lysis in Nonidet P-40 lysis buffer and preclearance as described above. Approximately 1.5 mg of protein was incubated with GST or GST-Raf-1 protein bound to glutathione-Sepharose CL-4B for 3-4 h at 4 °C. After incubation, the beads were collected by centrifugation and washed three times with lysis buffer and once with PBS, and the bound proteins were released by treating the beads twice with 10 mM reduced glutathione in 25 mM Tris-HCl (pH 8.0). The pooled fractions were mixed with 6× SDS sample buffer and boiled, and the proteins were resolved by SDS-PAGE and transferred to a PVDF membrane. The binding of Myc-MBS to GST-Raf-1 was detected by immunoblotting with monoclonal anti-Myc epitope antibody 9E10.

Far Western Blot/Overlay Assay-- COS-1 cells were transiently transfected with Myc-MBS or the Myc tag control vector (Myc) and lysed in radioimmune precipitation assay buffer (100 mM HEPES (pH 7.4), 10% glycerol, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 5 mM Na₃VO₄, 30 mM -glycerophosphate, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Protein (1.5 mg) was precleared with protein A/G-agarose for 1.5 h, and exogenous MBS was immunoprecipitated overnight with 3.5 µg of anti-Myc antibody 9E10. The immune complex was captured with protein A/G-agarose for 1.5 h, and immunoprecipitated Myc-MBS was resolved by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk in PBS/Tween for 3 h at 4 °C and incubated overnight at 4 °C with blocking buffer (PBS/Tween, 1.5% nonfat dry milk, 10 mM MnCl₂, 5 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM ATP, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) containing 0.5 mg/ml GST-Raf-1 fusion protein or GST (as a negative control). Bound GST-Raf-1 protein was observed by incubation of the membrane with 0.5 µg/ml anti-GST antibody for 1 h, followed by ECL detection. The membrane was reprobed with anti-Myc epitope antibody 9E10 to confirm the co-localization of Myc-MBS and GST-Raf-1.

MBS Phosphorylation Assay-- Purified GST-MBS protein was used as substrate in in vitro kinase reactions performed using purified GST-Raf-1 fusion protein or immunoprecipitated FLAG-tagged full-length Raf-1 as detailed below.

COS-1 cells were transiently transfected with FLAG-Raf-1 or FLAG-Raf-1(K375M). Thirty-six h after transfection, cells were serum-starved overnight and stimulated for 20 min with 200 nM phorbol 12-myristate 13-acetate (PMA) or vehicle (Me₂SO). Cells were washed twice with ice-cold PBS and lysed in Nonidet P-40 lysis buffer (with 200 mM NaCl). Lysates were centrifuged for 20 min at 16,000 × g. The supernatant (~1 mg of protein) was precleared with protein A/G-agarose for 2 h. Exogenous Raf-1 was immunoprecipitated overnight at 4 °C with monoclonal anti-FLAG antibody M2 (Sigma). The immune complex was captured by protein A/G-agarose for 1 h. The beads were washed three times with Nonidet P-40 lysis buffer and once with kinase buffer (50 mM HEPES (pH 7.4), 1 mM dithiothreitol, 10 mM MnCl₂, 5 mM MgCl₂, 1 µM okadaic acid, 15 µM ATP, and 10 µCi of [-³²P]ATP (6000 Ci/mmol)). An in vitro kinase assay was performed at 30 °C for 60 min in 30 µl of kinase buffer containing 2 µg of GST-MBS. The reaction was terminated by adding 6× SDS sample buffer and boiling for 4 min. The samples were electrophoresed on 7.5% SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was first stained with Ponceau S to visualize total GST-MBS (~55 kDa) and then exposed to x-ray film. After autoradiography, the membrane was immunoblotted with monoclonal anti-FLAG antibody M2 to visualize FLAG-Raf-1 (wild-type or K375M, ~74 kDa). The autoradiographs were scanned using the ImageQuant software program (Molecular Dynamics, Inc.). The arbitrary scanner values were plotted as -fold control (untransfected and untreated COS-1 cells).

For stoichiometric analysis of GST-MBS phosphorylation by Raf-1 kinase, FLAG-Raf-1 was immunoprecipitated from lysates of COS-1 transfectants (500 µg of protein) after PMA treatment as described above. The in vitro kinase reactions were carried out in parallel for various time points in kinase buffer containing FLAG-Raf-1 immune complex, 200 µM [-³²P]ATP (~900 cpm/pmol), and 40 µg/ml GST-MBS. The reactions were terminated by adding 6× SDS sample buffer and boiling for 4 min. The samples were electrophoresed on 7.5% SDS-polyacrylamide gel. The gel was stained with colloidal Coomassie Blue G-250 and dried, followed by autoradiography. The radioactive band (~55 kDa) corresponding to the phosphorylated substrate was excised, and radioactivity was determined in a scintillation counter.

In vitro phosphorylation of purified GST-MBS protein in the presence of purified GST-Raf-1 was performed using the kinase reaction conditions described above. In vitro phosphorylation of purified GST-MBS by catalytically active, recombinant Rho-associated kinase- (ROK; 66 kDa) (Upstate Biotechnology, Inc.) was performed exactly as described (15).

MBS Peptide Phosphorylation Assay-- Purified GST-Raf-1 or endogenous Raf-1 immunoprecipitated from MDA-MB 231 cells was used to phosphorylate an MBS-specific peptide, P3, representing amino acids 683-701 of the MBS protein (NH₂-KARSRQARQSRRSTQGVTL-COOH) (23). P3 containing a single amino acid mismatch, P3m (Thr⁶⁹⁶ to Val, NH₂-KARSRQARQSRRSVQGVTL-COOH), and two other MBS peptides, P1 (NH₂-VTTPTVSSGQATPTSPIK-COOH, amino acids 395-412) and P2 (NH₂-ISPKEEERKDESPATWRLGLRK-COOH, amino acids 421-442) (23), were also designed and tested. GST-Raf-N containing only the NH₂ terminus of Raf-1 was used as a negative control. Immunoprecipitated Raf-1 (~750 µg of total protein) or GST-Raf-1-Sepharose (20 ng) was incubated with the peptide (150 µM) for 15-20 min at 30 °C in 50 µl of the kinase reaction mixture, and peptide-associated radioactivity was quantified by liquid scintillation. Unless otherwise indicated, data were plotted using the radioactivity values obtained with P1 as a base-line control.

Sequential Kinase-Phosphatase Assay-- Raf-1 was immunoprecipitated from MDA-MB 231 cell lysates (2 mg of protein) prepared in Nonidet p-40 lysis buffer without the serine/threonine phosphatase inhibitors as described above. The beads were resuspended in 50 µl of the kinase reaction mixture containing 50 mM HEPES (pH 7.4), 12 mM MnCl₂, 1 mM dithiothreitol, and 20 or 100 µM nonradioactive ATPS (Sigma). The control reaction did not contain ATPS. In addition, a kinase reaction was also performed in the presence of an ROK inhibitor, HA-1077 (Calbiochem) or Y-27632 (Upstate Biotechnology, Inc.). The reaction was carried out at 30 °C for 30 min, followed by microcentrifugation. Phosphatase activity was assayed using the serine/threonine protein phosphatase assay system and phosphorylated myelin basic protein (MBP^P; ³³P-labeled MBP) according to the manufacturer's protocol (New England Biolabs Inc.). Phosphatase activity in Raf-1 immunoprecipitates was also assayed using MLC^P (4 µM, ³²P-labeled MLC) as substrate. The phosphatase reaction was carried out at 30 °C for 5 min (MLC^P) or 10 min (MBP^P). The radioactivity released in the supernatant was measured by liquid scintillation counting. The reaction was performed in duplicate per data point per experiment. Proteins in the pellet (Raf-1 immune complex, 1.0-1.5 mg of protein) were resolved by 7.5% SDS-PAGE, followed by sequential immunoblotting of the same blot with anti-MBS, anti-PP1, and anti-Raf-1 antibodies and ECL to detect MBS, PP1, and Raf-1 protein expression, respectively. The ECL signals were computer-scanned using ImageQuant software. The relative amounts of Raf-1-associated PP1 protein in various samples were determined by dividing the PP1 arbitrary scanner value by the Raf-1 value for that lane. Phosphatase-specific activity was then calculated using the following formula: absolute radioactivity (cpm)/relative amount of Raf-1-associated PP1 protein. The phosphatase-specific activity data were plotted as -fold base-line control reaction, i.e. without ATPS.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Physical Interaction of Raf-1 and MBS-- To identify new substrates of Raf-1 protein kinase, the whole cell lysates of MDA-MB 231 human breast carcinoma cells were examined for proteins associated with Raf-1. Two proteins (~130 and ~135 kDa) co-immunoprecipitated with Raf-1 and were readily visible on a Coomassie Blue-stained gel. These proteins were purified by sequential fractionation on SDS-polyacrylamide gel, followed by tandem mass spectrometric analysis. Four peptides from the 130-kDa band (peptides 1-4) and two peptides from the 135-kDa band (peptides 5 and 6) matched with MBS of human myosin phosphatase (Table I). In addition, three mass spectra from the 130-kDa band matched with myosin phosphatase protein from rat and chicken (data not shown).

To confirm the in vivo association of Raf-1 and MBS, immunoprecipitation and immunoblot experiments were performed. Co-immunoprecipitation of endogenous Raf-1 and MBS was observed in MDA-MB 231 cells (Fig. 1, A and B). To determine whether exogenous MBS interacts with Raf-1, COS-1 cells were transiently transfected with the expression vector containing Myc-tagged full-length MBS. The expression of Myc-MBS (~140 kDa) in COS-1 transfectants was verified by immunoblotting with anti-Myc epitope antibody (Fig. 1C). The interaction of Myc-MBS with GST-Raf-1 fusion protein (~100 kDa) (8) was observed by two independent approaches, the GST-Raf-1 pull-down assay (Fig. 1C) and the overlay assay (Fig. 1D). These data established a direct association between Raf-1 and MBS in MDA-MB 231 and COS-1 cells.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Raf-1 protein kinase associates with MBS in vivo and in vitro. A and B, reciprocal co-immunoprecipitation of endogenous Raf-1 and MBS proteins. Raf-1 or MBS was immunoprecipitated from lysates of log-phase MDA-MB 231 cells, followed by sequential immunoblotting (IB) of Raf-1 (A) and MBS (B) immunoprecipitates (IP) with anti-Raf-1 and anti-MBS antibodies, respectively. ML, mock lysate. C, in vitro binding assay. Lysates from COS-1 transfectants expressing Myc-tagged full-length MBS protein were mixed with GST-Raf-1-Sepharose CL-4B or GST-Sepharose CL-4B. Binding of Myc-MBS to GST-Raf-1 was detected by washing the beads, followed by 7.5% SDS-PAGE and immunoblotting with anti-Myc antibody. The presence of GST-Raf-1 in beads was confirmed in parallel by immunoblotting with anti-GST antibody. D, far Western blot assay. COS-1 cells were transiently transfected with Myc-tagged control vector (lanes 1) or Myc-tagged full-length MBS expression vector (lanes 2). Myc-MBS was immunoprecipitated from cell lysates with anti-Myc antibody, followed by SDS-PAGE and transfer to PVDF membrane. The membrane was overlaid with soluble GST-Raf-1 or GST protein as described under "Experimental Procedures." Binding of GST-Raf-1 to Myc-MBS was detected by immunoblotting the membrane with anti-GST antibody. The membrane was reprobed with anti-Myc antibody to detect Myc-MBS.

Phosphorylation of MBS by Raf-1 Protein Kinase-- To address that the physical association of Raf-1 and MBS means that Raf-1 phosphorylates MBS, in vitro kinase assays were performed using purified GST-tagged truncated MBS protein (amino acids 654-880, GST-MBS, ~55 kDa) as a substrate of Raf-1. This fragment of MBS includes an inhibitory phosphorylation site (Thr⁶⁹⁶) (27). In a direct in vitro kinase assay, followed by SDS-PAGE and autoradiography, GST-Raf-1 protein kinase was observed to phosphorylate GST-MBS (Fig. 2, left panels). As a positive control, the catalytic domain of purified ROK protein (66 kDa) was shown to phosphorylate GST-MBS (Fig. 2, right panels).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Raf-1 protein kinase phosphorylates GST-MBS fusion protein. Two µg of GST-MBS protein was incubated with [-32P]ATP in the presence of purified GST-Raf-1, GST, or the catalytic domain of ROK (66 kDa) as a positive control. This was followed by 7.5% SDS-PAGE, Coomassie Blue G-250 staining of the gel, and autoradiography for 50 min (GST-Raf-1 and GST) or 15 min (ROK).

Treatment of cells with the protein kinase C activator PMA has been shown to decrease Raf-1 mobility and to enhance Raf-1-associated serine/threonine kinase activity (24) (data not shown). We investigated whether PMA-activated Raf-1 is more efficient in phosphorylating GST-MBS protein. COS-1 cells were transiently transfected with the expression vector containing FLAG-tagged wild-type Raf-1 or FLAG-tagged Raf-1(K375M). Following PMA treatment, exogenous Raf-1 was immunoprecipitated with anti-FLAG antibody, and the immune complex was used in an in vitro kinase assay. Representative data demonstrate that a significantly higher phosphorylation of GST-MBS occurred with PMA-stimulated FLAG-Raf-1 compared with the unstimulated counterpart (~3.0-fold) (Fig. 3B, WT/PMA versus WT). The final stoichiometry of phosphorylation of GST-MBS using the FLAG-Raf-1 immune complex as a source of Raf-1 protein kinase was ~0.1 mol of phosphate/mol of substrate (Fig. 3C). The reason for the slight activity seen in the presence of FLAG-Raf-1(K375M) immunoprecipitates is unclear and may represent some background activity. However, PMA treatment had a negligible effect on mutant Raf-1 kinase activity (Fig. 3B, K375M versus K375M/PMA). In addition, as would be expected, phosphorylation of GST-MBS by wild-type Raf-1 was found to be significantly higher compared with mutant Raf-1 (3.96-fold) (Fig. 3B, WT/PMA versus K375M/PMA). Similar observations were made when we used His₆-MEK1 protein (4.44-fold), a known physiological substrate of Raf-1 (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Activation of exogenous Raf-1 protein kinase is associated with enhanced phosphorylation of GST-MBS. A, COS-1 transfectants expressing FLAG-tagged full-length Raf-1 (wild-type (WT)) were treated with 200 nM PMA for 20 min at 37 °C (+) or were left untreated (). As a control, COS-1 transfectants expressing FLAG-tagged Raf-1(K375M) were treated as described above (+) or left untreated (). Exogenous FLAG-Raf-1 was immunoprecipitated from various cell lysates (1 mg of protein) with anti-FLAG antibody M2. In vitro kinase reactions were performed using immunoprecipitates in the presence of [-32P]ATP and 2 µg of GST-MBS. Reaction mixtures were separated by 7.5% SDS-PAGE and transferred to a PVDF membrane. The membrane was stained with Ponceau S to visualize total GST-MBS, followed by autoradiography and then immunoblotting (IB) with anti-FLAG antibody M2. IgGH, heavy chain IgG. B, quantification of the representative data shown in A is presented. Relative GST-MBS phosphorylation levels after normalization with control, untransfected, and untreated COS-1 cells () are shown. C, shown is the representative stoichiometry of GST-MBS phosphorylation. FLAG-Raf-1 was immunoprecipitated from lysates of FLAG-tagged wild-type Raf-1 transfectants (500 µg of protein) following PMA treatment as described for A. In vitro kinase reactions were performed for the indicated times using FLAG-Raf-1 immunoprecipitates in the presence of [-32P]ATP and 1 µg of GST-MBS. The proteins were separated by SDS-PAGE. The gel was stained with Coomassie Blue G-250 to visualize GST-MBS and dried, and the radioactive bands corresponding to GST-MBS were excised and quantified by scintillation counting.

We next designed three MBS-specific peptides (23) designated as P1 (amino acids 395-412), P2 (amino acids 421-442), and P3 (amino acids 683-701) and tested whether these are the novel peptides phosphorylated by Raf-1. GST-Raf-1 specifically phosphorylated P3, but not P2 or P1 (P3 versus P1 or P2, ~8-fold) (Fig. 4A). Furthermore, P3 containing a single amino acid mismatch (P3m, Thr⁶⁹⁶ to Val) exhibited significantly diminished phosphorylation compared with P3 (~4-fold), and GST-Raf-N fusion protein (containing only the amino terminus of Raf-1) was far less effective in phosphorylating P3 compared with GST-Raf-1 (~4.0-fold) (Fig. 4A). ROK has been shown to cause phosphorylation of MBS (13). HA-1077 (100 µM), a chemical compound previously shown to inhibit ROK activity (25), did not inhibit Raf-1 kinase activity and Raf-1-mediated phosphorylation of P3 (data not shown). Cellular Raf-1 also phosphorylated MBS peptide P3 compared with control MBS peptide P1 (~6.0-fold), and phosphorylation of a single amino acid mutant peptide (P3m) was relatively less (~2.0-fold) (Fig. 4B). The presence of HA-1077 in the reaction mixture did not affect the level of P3 phosphorylation (Fig. 4B). Cellular ROK was also found to phosphorylate P3, and HA-1077 inhibited this mode of P3 phosphorylation (data not shown). From these observations, it appears that Thr⁶⁹⁶ of MBS is an important phosphorylation site for Raf-1 protein kinase, although the presence of additional site(s) in the P3 peptide cannot be ruled out.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Raf-1 phosphorylates an MBS-specific peptide. A, an in vitro kinase reaction was performed using GST-Raf-1-Sepharose (20 ng) and 150 µM MBS peptide P3 (amino acids 683-701). The radioactivity values obtained were normalized to control MBS peptide P1 (amino acids 395-412). The data shown represent two to three independent experiments (mean ± S.D.), each data point performed in triplicate per experiment. Other peptides used include P2, another MBS-specific peptide (amino acids 421-442), and P3m, peptide P3 with a single amino acid mismatch. GST-Raf-N is the fusion protein of GST and the Raf-1 amino terminus. B, Raf-1 was immunoprecipitated (IP) from lysates of logarithmically growing MDA-MB 231 cells (750 µg of protein), and an in vitro kinase reaction was performed in the presence of 150 µM P3, P1, or P3m and with or without ROK inhibitor HA-1077 (100 µM). Data shown represent one to three independent experiments, each data point performed in triplicate per experiment. C, mitotic Raf-1 was immunoprecipitated with monoclonal anti-Raf-1 antibody from MDA-MB 231 cells treated with nocodazole (40 ng/ml, 9 h). Raf-1 immunoprecipitates (750 µg of protein) were used in in vitro kinase reactions in the presence of 150 µM P3 or P2. Data shown are from a representative experiment, each data point performed in triplicate or quadruplicate (mean ± S.D.). ML, mock lysate.

Phosphorylation of Ser⁴³⁰ of chick MBS (which corresponds to Thr⁴³⁵ of human MBS) (26) during mitosis has been associated with activation of myosin phosphatase, and the P2 peptide contains Thr⁴³⁵. To further confirm Raf-1 selectivity for the P3 site, the phosphorylation of MBS peptides P3 and P2 was compared using Raf-1 immunoprecipitated from nocodazole-arrested MDA-MB 231 cells enriched in the mitotic phase (G₂/M > 93%). Consistent with the data shown in Fig. 4A, Raf-1 phosphorylated P3, but not P2 (Fig. 4C).

Inhibition of Raf-1-associated Protein Phosphatase-- We used a sequential kinase-phosphatase assay to unequivocally demonstrate the inhibition of Raf-1-associated myosin phosphatase activity. We used nonradioactive ATPS in the kinase reaction and MBP^P or MLC^P as substrate in the phosphatase reaction. Thio-phosphorylation of cellular MBS (co-immunoprecipitated with Raf-1) was performed because it is resistant to phosphatase activity of the myosin phosphatase holoenzyme (27). In the presence of ATPS (100 µM), endogenous protein phosphatase activity associated with Raf-1 immune complexes decreased by ~55 and ~70% with MBP^P and MLC^P as substrates, respectively (Fig. 5, A and B). Similar results were obtained when the ATPS concentration was reduced to 20 µM (data not shown). The presence of ROK inhibitors HA-1077 (100 µM) and Y-27632 (20 µM) (26) did not prevent reduction of phosphatase activity, establishing that the observed phosphorylation of MBS and inhibition of myosin phosphatase activity are not due to ROK.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Sequential kinase-phosphatase assay showing that Raf-1-associated protein phosphatase is inhibited. An in vitro kinase reaction was initiated using Raf-1 immunoprecipitated (IP) from logarithmically growing MDA-MB 231 cell lysates (2 mg of protein) and nonradioactive ATPS (100 µM) in the presence or absence of ROK inhibitor HA-1077 (100 µM) or Y-27632 (20 µM), followed by the phosphatase assay using MBPP (MyBPP; 33P-labeled MBP; A) or MLCP (32P-labeled MLC; B) as substrate. Control reactions were performed in the absence of nonradioactive ATPS. Phosphatase-specific activity was calculated based on the radioactivity value normalized against the level of Raf-1-associated PP1 protein determined by immunoblotting and quantification as explained under "Experimental Procedures." Data from representative experiments are shown.

Stimulation of Raf-1 and MBS Interaction by Ionizing Radiation and Concomitant Inhibition of Raf-1-associated Protein Phosphatase-- Previously, we demonstrated that exposure of human tumor cells (PCI-04A and MDA-MB 231) to ionizing radiation (IR) results in tyrosine phosphorylation, membrane translocation, and activation of Raf-1 (17, 18). To determine whether myosin phosphatase is a target of Raf-1 protein kinase in irradiated cells, we first examined the effect of IR on the association of MBS and PP1 with cellular Raf-1. IR treatment of MDA-MB 231 cells caused a significant increase in the association of Raf-1 with MBS (~3-6-fold) and PP1 (~3-fold) (Fig. 6, A-C). No change in the total amount of Raf-1, MBS, or PP1 protein per se was detected after irradiation (data not shown). These results suggest that activated Raf-1 selectively associates with myosin phosphatase.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Ionizing radiation stimulates interaction of Raf-1 with endogenous MBS and PP1 and inhibits Raf-1-associated protein phosphatase. A, MDA-MB 231 cells were irradiated (15 gray), followed by incubation for 2 h prior to cell lysis. Raf-1 immunoprecipitates (IP) were probed by serial immunoblotting (IB) with anti-MBS, anti-PP1, and anti-Raf-1 antibodies as described (17) (top). Quantification data (mean ± S.D.) from four to five independent experiments are shown (bottom). B, MBS was immunoprecipitated from whole cell lysates at ~15 min post-irradiation (15 gray), and immunoprecipitates were serially probed with anti-Raf-1 and anti-MBS antibodies. C, PP1 was immunoprecipitated from cell lysates within 2 h post-irradiation (15 gray) and serially probed with anti-Raf-1 and anti-PP1 antibodies. D, protein phosphatase-specific activity co-immunoprecipitating with Raf-1 was inhibited in irradiated MDA-MB 231 cells. Cell lysates were prepared at 2 h post-irradiation (15 gray). Raf-1 was immunoprecipitated with anti-Raf-1 antibody from cell lysates (1.0-1.5 mg of protein) prepared in Nonidet P-40 lysis buffer without the serine/threonine phosphatase inhibitors. Raf-1 immunoprecipitates were assayed for phosphatase activity using MBPP (MyBPP; 33P-labeled MBP) as substrate. Phosphatase-specific activity was calculated based on the radioactivity value normalized against the relative level of Raf-1-associated PP1 protein determined by immunoblotting of the Raf-1 immune complex and quantification as described in the legend to Fig. 5. Phosphatase-specific activities shown are from a representative of two independent experiments, each data point performed in duplicate per experiment.

We next measured protein phosphatase-specific activity in Raf-1 immune complexes from irradiated cells. As shown in Fig. 6D, protein phosphatase-specific activity in Raf-1 immunoprecipitates from irradiated cells was inhibited compared with unirradiated MDA-MB 231 cells (IR, 100%; +IR, ~54%). Additional metabolic labeling and immunoprecipitation experiments indicated that irradiation of MDA-MB 231 cells also led to a modest increase (~50%) in the total pool of phosphorylated MBS (data not shown). Consistent with the effects of okadaic acid on PP1 phosphatases (28, 29), phosphatase activity present in Raf-1 immunoprecipitates was not affected by 2 nM okadaic acid, but was inhibited by 2 µM okadaic acid (5 min at 30 °C) (data not shown). These data indicate a functional association of Raf-1 with PP1 protein phosphatase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Very limited information is available on the role of Raf-1 protein kinase in cytoskeletal reorganization. This study provides a direct link between Raf-1 and myosin phosphatase, an important component of pathways regulating cytoskeletal reorganization, cytokinesis, and cell motility. Rho kinase, a ZIP-like kinase, and myotonic dystrophy protein kinase have been shown to phosphorylate MBS at Thr⁶⁹⁶, resulting in the inhibition of myosin phosphatase (14, 15, 30, 31). Our findings concur with these observations in the sense that Raf-1 protein kinase targets Thr⁶⁹⁶ in MBS; we cannot, however, rule out the presence of other sites in MBS preferentially phosphorylated by Raf-1. Previously, MBS-specific peptide P2 has been shown to be phosphorylated (MBS Ser⁴³⁰) by a mitotic kinase, resulting in the activation of myosin phosphatase activity (26). Our present observations that the P2 peptide is not phosphorylated by Raf-1 emphasize the importance of Raf-1-specific inhibition of myosin phosphatase. In addition, Raf-1 does not phosphorylate another MBS-specific peptide, P1. Whether regulation of myosin phosphatase by Raf-1 can lead to a biological response distinct from other known and unknown MBS kinases remains to be seen. Our data appear to support the general notion that, depending on the cell type and stimulation, physiological compensatory mechanisms including regulation of myosin phosphatase activity are governed by overlapping and complementary pathways.

Dynamic reorganization of the actin cytoskeleton is an integral aspect of cellular responses to environmental signals. The small GTPase Rac is required for the formation of lamellipodia at the front of migrating cells, whereas at the rear of the cell, phosphorylation of MLC produces actomyosin contractility and de-adhesion necessary for cell movement (11, 12, 32, 33). Interestingly, Rac influences the cell migration process by selectively synergizing with Raf-1 kinase (6). In addition, COS-1 cells transiently transfected with the expression vector containing GST-Raf-1 demonstrate a significant increase in cell migration in collagen I.² Cell migration is an integrated process that depends on contractility of actomyosin microfilaments (11). Specific inhibition of myosin phosphatase in fibroblasts by the inhibitor protein CPI-17 causes overall shrinkage and contraction of the cells plus reorganization of the actin cytoskeleton with bundling of peripheral stress fibers and formation of membrane protrusions (34). Proteins that serve as membrane-cytoskeletal linkers, specifically moesin of the ERM family of proteins (35) and adducin (36), have been identified as potential substrates of myosin phosphatase that also bind to MBS (37, 38). The phosphorylation of these proteins needs to be sustained to extend filopodia, involving activation of another small GTPase, Cdc42 (39). An important role for activated Raf-1 at the membrane surface might be to locally inhibit myosin phosphatase activity to stabilize the cortical actin network and to enable extension of filopodia. This is consistent with the association of Raf-1 with plasma membrane-cytoskeletal elements and microfilaments (40-42). Furthermore, MBS has been localized in sites of cell-cell adhesion in epithelial cells, and myosin phosphatase regulation of protein phosphorylation at cell-cell contact sites has been suggested (43). Ionizing radiation modulates stress fiber formation in MDA-MB 231 cells.² Future studies will be designed to examine the role of myosin phosphatase in radiation-related modifications of the cytoskeleton. In summary, our report provides evidence for a signaling pathway in which activated Raf-1 targets MBS and inhibits the interacting protein phosphatase under physiological and stress-related conditions. This offers another way for signaling pathways to connect to cytoskeletal reorganization that underlies changes in cell shape and cell motility.

	ACKNOWLEDGEMENTS

We thank Dr. D. A. Cheresh for discussions and Dr. A. Dritschilo for support. The cell cycle analysis was performed at the FACS Resource of the Lombardi Cancer Center.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants CA68322 (to U. K.), Grants CA40042 and GM56362 (to D. L. B.), and Program Project Grant CA74175 and by an American Heart Association Affiliate postdoctoral fellowship award (to M. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Lombardi Cancer Center, Georgetown University Medical Center, E208 Research Bldg., 3970 Reservoir Rd. NW, Washington, D. C. 20007. Tel.: 202-687-2226; Fax: 202-687-0400; E-mail: kasidu@georgetown.edu.

Published, JBC Papers in Press, November 21, 2001, DOI 10.1074/jbc.M106343200

² C. G. Broustas, and U. Kasid, unpublished data.

	ABBREVIATIONS

The abbreviations used are: MBS, myosin-binding subunit; PP1, protein phosphatase-1; MLC^P, phosphorylated myosin light chain; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; PMA, phorbol 12-myristate 13-acetate; ROK, Rho-associated kinase-; ATPS, adenosine 5'-O-(3-thiotriphosphate); MBP^P, phosphorylated myelin basic protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; IR, ionizing radiation.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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