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J. Biol. Chem., Vol. 281, Issue 42, 31202-31211, October 20, 2006

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Phosphorylation of Myosin Phosphatase Targeting Subunit 3 (MYPT3) and Regulation of Protein Phosphatase 1 by Protein Kinase A^*

Jeffery Yong, Ivan Tan, Louis Lim, and Thomas Leung¹

From the GSK-IMCB Group, Institute of Molecular and Cell Biology, Singapore 138673, Singapore and the Department of Molecular Neuroscience, Institute of Neurology, University College London, 1 Wakefield Street, London WC1N 1PJ, United Kingdom

Received for publication, August 1, 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Myosin phosphatase targeting subunit 3 (MYPT3) and transforming growth factor--inhibited membrane-associated protein (TIMAP) are two closely related myosin-binding targeting subunits of protein phosphatase 1 (PP1c) with a characteristic CAAX (where AA indicates aliphatic amino acid) box at the C termini. Here we show that MYPT3 can be a substrate for protein kinase A (PKA). We first mapped the multiple phosphorylation sites within a central conserved motif. Deletion or mutations of this motif resulted in enhancement of the associated PP1c activity, suggesting that phosphorylation of MYPT3 may play an important role in regulating PP1c catalytic activity. However, unlike the other known MYPTs, which upon phosphorylation inhibit PP1c, PKA phosphorylation of MYPT3 resulted in PP1c activation, indicating a different mode of action. There is a direct interaction between the central conserved phosphorylated site motif with the N-terminal ankyrin repeat region; this interaction was significantly reduced with MYPT3 phosphorylation or acidic phosphorylation site mutations, with concomitant alterations in biochemical and morphological consequences. We therefore propose a novel mechanism for the phosphorylation of MYPT3 by PKA and activation of the catalytic activity through direct interaction of a central region of MYPT3 with its N-terminal region.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In mammalian cells, the phosphorylation states of the regulatory light chain of myosin (RMLC)² are key determinants of the myosin motor in driving inter- and intracellular movement, muscle contraction, and cell shape regulation (1, 2). Myosin light chain kinase and phosphatase (MLCP) are key enzymes in this regulation and are therefore critical in the maintenance of cell contraction and relaxation as well as cell morphology (3, 4). However, there is growing evidence that Rho kinase (ROK) may also regulate MLCP and play an important role alongside myosin light chain kinase in regulating phosphorylation of RMLC (4, 5). Conventional MLCPs consist of a catalytic protein phosphatase 1 (PP1c) subunit, a myosin phosphatase targeting subunit (MYPT), and a smaller M20 subunit, although not always present in all the phosphatase complexes (6–8). PP1c, like most serine/threonine phosphatases, is widely nonspecific and requires proper localization and targeting for efficient dephosphorylation of its substrates, usually bestowed by a targeting subunit (9). Accordingly, studies have shown that the MYPT subunit is essential for myosin phosphatase activity, as there is a significant reduction in the ability of the PP1c alone to dephosphorylate substrate phospho-RMLC (p-RMLC) (10, 11).

A typical MYPT subunit consists of a short N-terminal PP1c-binding RVXF motif followed by multiple ankyrin repeats that are required for substrate specificity and may also be partly for targeting. The C-terminal one-third of MYPT1 has been reported to interact with RhoA, protein kinase G, phospholipids, and myosin (11–13). However, the N-terminal PP1c binding and ankyrin repeats of MYPT1 have also been shown to be sufficient for efficient targeting of PP1c to phospho-RMLC (10, 11). The crystal structure of the N-terminal MYPT1-PP1c complex also suggests that apart from the RVXF motif, there are also considerable weak contacts between the catalytic subunit and the ankyrin repeats of MYPT1 (14). These analyses also reveal that such interactions alter the shape of the catalytic cleft and charge distribution that may determine the substrate entry, and hence the targeting specificity.

MYPT1, MYPT2, and the closely related MBS85 also play a regulatory role for the myosin phosphatase holoenzyme. There are several putative phosphorylation sites on these isoforms of the MYPT family, and some had been shown to be phosphorylated and regulated by a range of kinases, including Rho kinase (ROK/ROCK), myotonic dystrophy-related CDC42-binding kinase (MRCK), myotonic dystrophy protein kinase, Raf-1, and integrin-linked kinase (12, 15–19). Phosphorylation of a threonine in MYPT1 (Thr-696) and MBS85 (Thr-560) in a highly conserved motif resulted in inhibition of PP1c activity toward p-RMLC. Recently, it was reported that phosphorylation of an adjacent serine residue (Ser-695) in MYPT1 prevented the phosphorylation of Thr-696, adding yet more complexity to the regulation of MYPT1 (20). Apart from these known regulatory subunits, there are several other members of a growing MYPT protein family, including the unconventional myosin Myr8 isoforms (21) and the closely related MYPT3 and TIMAP (22–24). However, it is not known if these novel MYPT-related proteins are bona fide targeting subunits of myosin phosphatase with similar phosphorylation regulation mechanisms.

Interestingly, MYPT3 and TIMAP share similar domain structures that differ from all other MYPT family members (7, 23, 24). Both have a C-terminal CAAX prenylation motif that targets the protein to cell membranes, similar to that of the Rho GTPases (25). Considering reports that the localization of myosin phosphatase may have a role in its regulation and function (26), it is possible that there are specialized enzyme complexes that localize to the cell membranes and may affect actin structures at the extreme cell periphery or barrier function in endothelial cells.

Surprisingly, MYPT3 was recently reported to be inhibitory toward PP1c dephosphorylation of p-RMLC (22), although it has features shared with the other well studied MYPT family members, particularly the PP1c-binding site and the ankyrin repeats region at the N terminus. As these conserved domains alone were shown to be sufficient for myosin phosphatase targeting function (10, 11), it is unusual for MYPT3 not to have any positive effects on the catalytic activity. Furthermore, Drosophila MYPT-75D, an ortholog of mammalian MYPT3, has been shown recently to be able to increase PP1 activity toward phospho-Sqh (Drosophila p-RMLC) (27). These observations have suggested to us that mammalian MYPT3 may also have effects on PP1c activity and RMLC phosphorylation but may be under stringent regulation. Both MYPT3 and TIMAP contain several putative phosphorylation sites in a highly conserved central region. We therefore speculated that a previously unidentified kinase could phosphorylate MYPT3 and possibly regulate the phosphatase activity. Here we report that MYPT3 can be a substrate of protein kinase A and that phosphorylation resulted in disruption of an interaction between the central phosphorylation site motif and the ankyrin repeat region, resulting in PP1c activation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture and Immunocytochemistry—HeLa cells were maintained in minimum Eagle's medium supplemented with 10% fetal bovine serum, 2 mML-glutamine, 10 mM sodium pyruvate, 0.15% w/v sodium bicarbonate, and 0.1 mM minimum Eagle's medium nonessential amino acids (Invitrogen) and were plated on glass coverslips at 80% confluence for transfection. Cells were transfected with various constructs using Lipofectamine (Invitrogen) according to the manufacturer's instructions. After fixing with 4% paraformaldehyde, cells were co-stained with anti-FLAG or HA antibodies and either TRITC-conjugated phalloidin (Sigma) for polymerized actin or anti-phosphomyosin light chain 2 (Ser-19) (p-RMLC) antibodies (Cell Signaling Technology). Cells were treated with either 40 µM forskolin (Sigma) or Me₂SO for 45 min before fixation in 4% paraformaldehyde.

DNA Constructs—Full-length MYPT3 (FL) (residues 1–528) was obtained by joining two human EST clones (BE281125 [GenBank] and BG422824 [GenBank] ) at the SmaI site. The 1.7-kbp fragment was obtained by PCR with primer pair 5'-CAGGATCCATGGCCGAGCACCTGG-3' and T7, and the BamHI/BglII-digested fragment was cloned into pXJ40 FLAG, pXJ40 His₆, and pGEX 4T1 vectors. CAAX construct (residues 1–524) was obtained by PCR using the reverse primer 5'-CACTCGAGTCAGCACGGCCTCCTCTC-3' and the vector forward T7 primer. N-terminal construct (residues 1–395) was obtained by digestion with BamHI/SacI and ligation into pXJ40-FLAG vector. The ankyrin repeats, including the N-terminal PP1c-binding site (residues 1–324), was obtained by PCR using the reverse primer 5'-CAAAGCTTGGCGTCGTGCTTGTGCT-3' and the T7 primer for the pXJ40 vector. The C-terminal construct (residues 356–528) was obtained by PCR using forward primer 5'-CAAAGCTTCAGCGCACCGACCTGTAC-3' and reverse primer for the pXJ40 vector. The construct deleted of the conserved phosphorylation site (PS; deleted residues 325–355) was obtained by joining the AR and C-terminal constructs above via the HindIII site. PSCAAX construct was obtained by digesting PS and CAAX constructs with BamHI/SacI and ligation of the insert from PS into the vector of CAAX. Phosphorylation site construct (PS; residues 313–395) was obtained by PCR using forward primer 5'-CAGGATCCAAGCTGCTGGAGCTGAAGC-3' with the reverse primer for the pXJ40 vector with MYPT3-NT construct as the template. QuikChange II site-directed mutagenesis kit (Stratagene) was used to generate full-length S340A/S341A (RRTAA) and S353A (RRVA) mutant constructs from full-length MYPT3 using primer pairs 5'-CGCCGCACCGCCGCCGCCGGCAGC-3'/5'-GCTGCCGGCGGCGGCGGTGCGGCG-3' and 5'-GTGAGGCGGGTGGCCCTAACCCAGCGC-3'/5'-GCGCTGGGTTAGGGCCACCCGCCTCAC-3', respectively. S340A/S341A/S353A (3A) triple mutant construct was obtained using the full-length RRTAA mutant as template and the primer pair for generating the RRVA mutant from above. PS fragments of these mutants were obtained by PCR using the PS forward primer and the reverse primer for the vector, as with the wild type. PS-S340A mutant was obtained by using primer pairs 5'-GCCAGCGCCGGCAGCCGC-3'/R2 and 5'-GGTGCGCGGCGGCGCAGC-3'/T7 in a two-step PCR mutation as described previously (28). PS S341A was obtained similarly using primer pairs 5'-GCCGCCGGCAGCCGCGGG-3'/R2 and 5'-GGAGGTGCCGGCGGCGCAG-3'/T7. MYPT3-3D mutant constructs were also obtained via QuikChange II mutagenesis kit, using primer pairs 5'-GCCGCCGCACCGACGACGCCGGCAGCCG-3' and 5'-CGGCTGCCGGCGTCGTCGGTGCGGCGGC-3' for S340D/S341D and 5'-GGTGAGGCGGGTGGACCTAACCCAGCGCA-3' and 5'-TGCGCTGGGTTAGGTCCACCCGCCTCACC-3' for S353D on the S340D/S341D template. The PKA construct was obtained by PCR using primers 5'-CAGGATCCATGGGCAACGCCGCCG-3'/5'-CACTCGAGAACTCAGTAAACTCCTTGCC-3' and rat liver cDNA as template. The PCR product was then cloned into pXJ40 Myc and pGEX 4T1 via BamHI and XhoI sites for expression.

Gel Filtration Chromatography—COS7 cells were co-transfected with FLAG-CAAX and HA-PP1c and harvested in low salt buffer (50 mM HEPES, pH 7.3, 50 mM NaCl, 1x Complete protease inhibitor mixture (Roche Applied Science), 5% glycerol), homogenized, and centrifuged at 100,000 x g at 4 °C for 30 min to remove particulate matter and membranes. 100 µgof cleared lysate was applied to a PC 3.2/30 Superdex 200 column (Amersham Biosciences) equilibrated with the same buffer in a SMART system (Amersham Biosciences) at a flow rate of 50 µl/min. 25-µl fractions were collected, resolved on SDS-PAGE, and bands analyzed by Western blot. Bacterially expressed recombinant AR was purified and analyzed in the same buffer and column as described above.

In-gel Kinase Assay—0.5 mg/ml GST fusion protein of full-length MYPT3, or GST as control, was used as substrate cross-linked in the SDS-polyacrylamide gel matrix. After renaturation and equilibration as described elsewhere (29), the gels were cut into strips and incubated in in-gel kinase assay buffer (50 mM HEPES, pH 7.3, 5 mM MgCl₂, 5 mM MnCl₂, 0.1% Triton X-100) with 10 µM [-³²P]ATP and either 1 mM of the various kinase inhibitors or Me₂SO as control. The reactions were stopped by fixing in 40% methanol (v/v), 5% acetic acid (v/v), and stained with Coomassie Blue. Gel pieces were then dried and autoradiographed.

Immunoprecipitation—COS7 cells were plated at 70% confluence at the time of transfection with various DNA constructs and were lysed in immunoprecipitation (IP) buffer (containing 25 mM HEPES, pH 7.3, 150 mM NaCl, 0.2 mM EDTA, 1 mM Na₃VO₄, 1.5 mM MgCl₂, 20 mM -glycerol phosphate, 5% glycerol, 0.2% Triton X-100, and 1x protease inhibitor mixture) and centrifuged at 14,000 rpm at 4 °C for 10 min. Cleared lysates were incubated with anti-FLAG M2-agarose beads (Sigma) or nickel nitrilotriacetic acid-agarose (Qiagen) for 2 h at 4 °C before washing extensively with IP buffer. For His₆-tagged MYPT3 purification, 50 mM imidazole was added to the wash buffer, and the purified protein was eluted with 200 mM imidazole in IP buffer. For the analysis of FLAG-AR and GST-PS interaction, the NaCl and Triton X-100 concentrations were increased to 0.3 M and 1%, respectively, to reduce nonspecific binding.

In Vitro Phosphorylation Assays—His₆-tagged MYPT3 purified from COS7 cells was incubated with 10 µM [-³²P]ATP at 30 °C for 30 min before boiling in 2x SDS-PAGE sample buffer. For PKA kinase assays, 0.5 units of bovine heart PKA (Sigma) or 0.5 µg of GST-PKA was added together with 10 µM [-³²P]ATP in PKA buffer (50 mM Tris-HCl, pH 7.4, 20 mM MgCl₂, and 1 mM dithiothreitol), and the reaction was carried out at 30 °C for 30 or 60 min. The reactions were stopped by boiling in 2x SDS-PAGE sample buffer, separated on SDS-PAGE, transferred to polyvinylidene difluoride membrane (PerkinElmer Life Sciences), and autoradiographed.

PKA Phosphorylation of MYPT3 in Intact Cells—HeLa cells expressing FLAG-MYPT3 and HA-PP1c were treated with 40 µM forskolin, 40 µM H89 or Me₂SO for 3 h or were co-transfected with Myc-tagged PKA, harvested in 2x SDS-PAGE sample buffer supplemented with 100 nM okadaic acid and 100 nM calyculin A (both from Alomone Labs), and boiled for 15 min. Samples were then diluted with three volumes of IP buffer before being subjected to FLAG immunoprecipitation. All samples were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes for Western blotting with rabbit monoclonal anti-phospho-PKA substrate antibody (Cell Signaling Technology).

Myosin Light Chain Phosphatase Assays—The coding sequence of the non-muscle regulatory myosin light chain was obtained by PCR from HeLa cell cDNA and subcloned into pGEX-4T1 for bacterial expression as described (19). ³²P-Phosphorylated MLC2 protein was obtained by phosphorylation with MRCK catalytic domain (MRCK-CAT) in 10 µM ATP with 10 µCi of [-³²P]ATP as described previously (19). COS7 cells were transfected with FLAG-tagged MYPT3 and HA-tagged PP1c and immunoprecipitated using anti-FLAG M2 beads in MBS purification buffer (25 mM HEPES, pH 7.3, 150 mM NaCl, 0.5 mM EDTA, 1 mM MgCl₂, 5% glycerol, 0.2% Triton X-100), and the resulting purified complex was incubated with 10 µgof ³²P-RMLC in phosphatase assay buffer (50 mM HEPES, pH 7.3, 50 mM NaCl, 1 mM MnCl₂, 0.1% Triton, 2 mM dithiothreitol) at 30 °C. At 0, 15, 30, and 45 min, a fraction of the reaction was removed and stopped by boiling in 2x SDS-PAGE sample buffer and dotted onto nitrocellulose membranes. Membranes were then washed extensively with 10 mM phosphoric acid, dried, and analyzed on a Molecular FX Imager (Bio-Rad) using a Kodak Storage PhosphorScreen (Amersham Biosciences). For PKA-phosphorylated MYPT3, immunoprecipitated complexes were phosphorylated with and without 0.1 µg of GST-PKA (cloned from rat liver cDNA) fusion protein in 50 mM Tris-HCl, pH 7.4, 20 mM MgCl₂, 0.1 mM ATPS, and 1 mM dithiothreitol for 60 min. Complexes were then washed extensively with MBS purification buffer before proceeding with the phosphatase assays. 2 µg of the purified bacterially produced GST-PS protein was phosphorylated with and without 0.2 µg of GST-PKA and ATPS in PKA buffer for 60 min before being added to the phosphatase assays.

Two-dimensional Tryptic Peptide Mapping—GST-PS fusion proteins were produced and purified by affinity chromatography, and the GST tag was removed by cleavage with thrombin. 5 µg of the various untagged PS fusion proteins were phosphorylated by 1 µg of GST-PKA with 0.1 mM ATP and 10 µCi of [-³²P]ATP for an hour at 30 °C before digestion with trypsin as described (30). Briefly, the ³²P-phosphorylated proteins were separated on SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The radioactive protein bands were excised and digested with 1 unit of trypsin (Roche Applied Science) per µg of protein substrate for 4 h at 37 °C, and peptides were recovered from the supernatant. Concentrated peptides were then spotted on 20 x 20-cm microcrystalline cellulose TLC plates (Eastman Kodak Co.), coated with buffer (2.2% (v/v) formic acid, 7.8% (v/v) acetic acid), and electrophoresis carried out at 200 V at 10 °C for 3 h. Plates were then air-dried and separated by TLC for the second dimension in phosphopeptide chromatography buffer (butanol-1/pyridine/acetic acid/water (75: 50:15:60, v/v)), after which the plates were thoroughly dried and autoradiographed.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Phosphorylation of MYPT3 by PKA—Purified His₆-tagged 62-kDa MYPT3 protein from COS7 cells overexpressing MYPT3 was found to be phosphorylated by an endogenous kinase(s) (Fig. 1A). We therefore tested several candidate kinases for MYPT3 phosphorylation. ROK and MRCK, previously shown to phosphorylate other members of the MYPT family (12, 19), were unable to phosphorylate MYPT3 in vitro (data not shown). Therefore, an in-gel kinase assay was used to detect any possible renaturable kinases that could phosphorylate MYPT3. As shown in Fig. 1B, a common 40-kDa kinase band was observed in the GST-MYPT3 gel for several tissue and cell lysates but not in the GST control gel. We speculated that the putative kinase could be the catalytic subunit of PKA. We then modified the in-gel kinase assay to include kinase inhibitors, H-7, H-89, HA-1077, and Me₂SO as a control. The kinase was largely inhibited by the addition of H-89, a known inhibitor of PKA. Control purified bovine PKA catalytic subunit was also similarly inhibited (Fig. 1C), suggesting that the unknown kinase may be the catalytic subunit of PKA.

View larger version (44K): [in this window] [in a new window]   FIGURE 1. In-gel kinase assay detection of MYPT3 kinase. A, overexpressed MYPT3 can be phosphorylated by endogenous kinase(s). His6-tagged MYPT3 was purified from COS7 cells, and 10 µM [-32P]ATP was added and incubated at 30 °C for 30 min. The proteins were separated on SDS-PAGE, stained with GelCode Blue (Pierce), and dried for overnight autoradiography. C denotes the control lane (beads with untransfected cell lysate), and the MYPT3 band is indicated by arrowheads. A co-precipitated 37-kDa protein, marked by an asterisk, was identified as PP1c by mass spectrometry. B, in-gel kinase assays using GST or GST-MYPT3 cross-linked into the gel as substrates. 50 µg of lysates were loaded onto each lane. Arrowhead points to a 40-kDa band observed in the GST-MYPT3 gel autoradiograph not present in the GST gel. C, effects of various kinase inhibitors. In-gel kinase assays of rat brain lysate or 0.5 units of bovine PKA were carried out in the presence of 1 mM kinase inhibitor or Me2SO (DMSO). D, phosphorylation of MYPT3 in intact cells. In vitro phosphorylation of MYPT3 was carried out with 1 µg of bacterially expressed GST-MYPT3 with and without GST-PKA and 1 mM ATP (left panel). For in vivo phosphorylation of MYPT3, HeLa cells were co-transfected with FLAG-MYPT3 and HA-PP1c constructs with or without Myc-PKA and treated as indicated. Proteins were separated on SDS-PAGE and blotted for phosphoprotein detection using a rabbit anti-phospho-PKA substrate antibody (Cell Signaling Technology); left panel shows IP products. Arrowheads indicate positions of the full-length MYPT3 proteins; asterisks indicate endogenous proteins that are phosphorylated by PKA, and white asterisks indicate breakdown products of the bacterial GST fusion protein.

Mapping of PKA Phosphorylation Sites—MYPT3 shares high homology with TIMAP, both having ankyrin repeats (AR) at the N terminus and a CAAX motif at the C terminus. A central conserved region in both MYPT3 and TIMAP also contains several putative phosphorylation sites (Fig. 2C). To confirm PKA phosphorylation of MYPT3 and to map the potential PKA phosphorylation site(s), we generated constructs expressing various FLAG-tagged fragments of MYPT3 (Fig. 2A). When these constructs were overexpressed together with HA-PP1c in COS7 cells and immunoprecipitated, it was found that full-length MYPT3 and fragments containing the central conserved motif were highly phosphorylated by PKA. The conserved motif that was phosphorylated is then designated as the phosphorylation site motif or PS (Fig. 2, A and B). To confirm that the phosphorylation sites were within this PS region, three suspected sites were mutated to deduce the phosphorylation site(s) of PKA. Mutation of the three serine residues at 340, 341, and 353 to alanine (PS 3A) reduced most of the phosphorylation by PKA, suggesting that these are the major sites in MYPT3 that can be phosphorylated by PKA (Fig. 2C). From the kinetic analysis, PS can be efficiently phosphorylated up to 1.2 mol of phosphate per mol of PS protein (Fig. 2D). Two-dimensional tryptic peptide mapping of phosphorylated PS mutants suggested that there is a preference of PKA for the sites Ser-340 and Ser-353, with an estimated ratio of phosphorylation of about 1:2, indicating that there is a higher preference for Ser-353 by PKA (Fig. 2E).

View larger version (68K): [in this window] [in a new window]   FIGURE 2. Mapping of PKA phosphorylation sites on MYPT3. A, diagrammatic representation of MYPT3 fragments used in this study. FL, full-length; CAAX, deleted CAAX; NT, N-terminus; AR, ankyrin repeats; CT, C-terminus; PS, phosphorylation site; PS, deleted phosphorylation site. B, identification of phosphorylation sites in PS fragment. Immunoprecipitated FLAG-tagged proteins expressed in cells were phosphorylated without and with 0.5 units of bovine heart PKA for 30 min, separated, and autoradiographed. MYPT3 protein fragments are indicated with asterisks. C, mapping of PKA phosphorylation sites within the PS fragment. The conserved regions of MYPT3 and TIMAP containing the potential phosphorylation sites are indicated. Serine to alanine mutations were obtained and expressed as GST fusion proteins. 5 µg of each fusion protein were subjected to PKA phosphorylation, resolved on SDS-PAGE, and autoradiographed. D, rate of recombinant PKA phosphorylation of PS and PS 3A. Two µg of PS protein were phosphorylated by 0.2 µg of GST-PKA in 0.1 mM [-32P]ATP, for 0, 1, 2, 5, 10, or 20 min, and phosphate incorporation was measured via scintillation counting. E, two-dimensional tryptic peptide mapping of PS fragments. Phosphorylated PS, PS single alanine mutants (PS 340A, PS 341A, and PS 353A), and PS 3A proteins were subject to tryptic digestion, two-dimensional thin layer chromatography, and autoradiographed.

MYPT3 Mutants Reveal Morphological Effects on Actin Cytoskeleton—To determine the importance of MYPT3 phosphorylation, we asked whether this isoform, like the other characterized isoforms, could regulate PP1c catalytic activity by post-translational modifications. We speculated that MYPT3 may have regulatory effects on PP1c catalytic activity, and this depends on the phosphorylation state of the central PS. To test this hypothesis, we first examined the phenotypic effects of the various mutants on disassembly of actin stress fibers, demonstrated previously to be a convenient way to show enhancement of PP1c activity (19, 28) as well as direct p-RMLC staining to look at actomyosin assembly. On expression in HeLa cells, both wild type (Fig. 4, a–c) and CAAX motif deletion mutant (Fig. 4, d–f) had no obvious effects on polymerized actin or filamentous p-RMLC staining, although localization of MYPT3 to the cell membrane was lost after CAAX deletion. When MYPT3 was truncated up to the ankyrin repeats we detected a decrease in p-RMLC staining, suggesting an increase in myosin light chain phosphatase activity (see also Fig. 6B, AR) as well as a loss of stress fibers (Fig. 4, g–i). These observations suggest that the region encompassing the PS may contain a regulatory motif for PP1c activation. To confirm this hypothesis, mutants deleted of this site, PS and PSCAAX, were used. The results (Fig. 4, j–o) showed that activities similar to those of the C-terminal truncated MYPT3(AR) were obtained, indicating that indeed the PS contributes to negative regulation on catalytic activity. It was also noted that the PS deletion mutant (PS, Fig. 4, j–l) had obvious enhanced effects on peripheral actin fibers, whereas a mutant further deleted of CAAX showed a general loss of stress fibers (PSCAAX, Fig. 4, m–o). These effects were most likely due to the membrane localization of wild type MYPT3 and activation at the membrane, as the removal of the CAAX motif resulted in cytoplasmic localization and a general loss of actomyosin networks within the cell.

View larger version (67K): [in this window] [in a new window]   FIGURE 3. MYPT3 and PP1c associated in dimeric complex. A, gel filtration analysis of FLAG-CAAX/HA-PP1c complex. Cleared lysate from COS7 cells transfected with FLAG-CAAX and HA-PP1c was fractionated with a Superdex 200 gel filtration column. Fractions were collected for Western blotting with anti-FLAG (MYPT3) and anti-HA antibodies (PP1c). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal marker, and standard external markers were used for plotting molecular sizes of the components (arrows). B, various FLAG-tagged MYPT3 fragments were co-expressed with HA-tagged MYPT3 fragments and Myc-tagged PP1c. Immunoprecipitations were performed with anti-FLAG antibody, and bound proteins were resolved on SDS-PAGE and analyzed by Western blotting.

To verify these observations, we further investigated the cellular morphological consequence of these mutants in HeLa cells and the effects of forskolin treatment. CAAX proteins were used as they produced a more general and therefore more easily scored effect on morphology compared with only the cell edges for the FL proteins. HeLa cells expressing FLAG-CAAX/HA-PP1c showed moderate loss of filamentous p-RMLC staining compared with neighboring untransfected cells, and forskolin treatment significantly increased the transfected cell population with filamentous p-RMLC loss (Fig. 5, B and C). As expected, cells transfected with FLAG-CAAX 3A/HA-PP1c elicited no such reduction when compared with HA-PP1c alone. In agreement with the biochemical data, the CAAX 3D mutant produced a constitutive reduction of p-RMLC staining when co-expressed with PP1c. Furthermore, unlike CAAX, both CAAX 3A and 3D mutants were insensitive to forskolin treatment. From the biochemical and morphological analyses, we conclude that phosphorylation of MYPT3 on PS could activate PP1c activity toward p-RMLC.

Regulated Interaction of Phosphorylation Site Motif with N-terminal Ankyrin Repeat Region—To probe the mechanism of how PS phosphorylation leads to an activation of the associated PP1c, we examined whether there were interactions between the different domains of MYPT3. As shown in Fig. 6A, GST-PS and GST-PS 3A both interacted with the N-terminal ankyrin repeat region (FLAG-AR). In contrast, the GST-PS 3D bound the AR with decreased affinity. The PKA-phosphorylated GST-PS also bound significantly less AR compared with unphosphorylated GST-PS. However, single aspartate mutants did not show significant differences in FLAG-AR binding from GST-PS protein (data not shown), suggesting that multiple phosphorylations may be required for a maximal effect. To test the significance of the interaction between the AR and the PS, phosphatase assays were conducted with the constitutively active FLAG-AR/HA-PP1c complex without and with bacterially produced PS-, PS 3A-, PS 3D-, or PKA-phosphorylated PS proteins. Interestingly, PS and PS 3A were able to inhibit the myosin light chain phosphatase activity of the FLAG-AR/HA-PP1c complex, and this inhibition was significantly diminished after PKA phosphorylation or acidic mutations of PS (Fig. 6B). PKA phosphorylation of the PS 3A mutant, however, did not alter its ability to inhibit the FLAG-AR/HA-PP1c activity (data not shown), giving further support that these PKA sites are solely responsible for the effects.

To determine the cellular significance of this interaction, we examined the effects of co-transfection of the HA-AR/Myc-PP1c complex with either wild type or mutant PS constructs for morphological analysis. As shown in Fig. 6, C and D, cells expressing HA-AR/Myc-PP1c alone showed a complete loss of actin filaments, but this did not occur when HA-AR/Myc-PP1c was co-expressed with either FLAG-PS or FLAG-PS 3A. In contrast, the binding-defective FLAG-PS 3D was least effective. Mutant proteins with single serine to aspartate mutations gave similar effects as FLAG-PS protein (data not shown) when expressed with HA-AR/Myc-PP1c. These results provide further evidence that unphosphorylated MYPT3, with an interaction between the PS with its N-terminal AR region, can exert an inhibitory effect on PP1c activity and that multiple phosphorylation or acidic mutations of the PS prevents such an interaction, resulting in PP1c activation (Fig. 7).

View larger version (129K): [in this window] [in a new window]   FIGURE 4. Morphological effects of mutants of MYPT3/PP1c in HeLa cells. HeLa cells were transfected with the various FLAG-MYPT3 constructs with HA-PP1c and co-stained with monoclonal anti-FLAG antibody, rabbit anti-phospho-RMLC, and phalloidin to show actin stress fibers. Transfected cells are marked with asterisks. Bar = 10 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Previously, MYPT3 in complex with PP1c was shown to exhibit inhibitory effects on PP1c dephosphorylation of both phosphorylated phosphorylase a and p-RMLC (22). However Drosophila MYPT-75D, an ortholog of MYPT3, was reported to have myosin light chain phosphatase activity in vitro and in vivo (42), suggesting to us that the mammalian counterpart may have a similar role in regulating actin-myosin events. We have reported previously that the disruption of actin cytoskeleton by active myosin phosphatase can be a good indication of the catalytic activity (19, 28). By using this cell-based assay, the dimeric MYPT3 in complex with PP1c was found to be relatively inactive but could be activated by a C-terminal truncation, suggesting that MYPT3 could indeed play a regulatory role for PP1c activation.

Both the characterization of MYPT3 phosphorylation by PKA and the morphological analysis of MYPT3 mutants also provided complementary evidence that a phosphorylation event on MYPT3 may regulate PP1c activity. Co-expression of a MYPT3 mutant devoid of the PS region together with PP1c led to the loss of both peripheral actin stress fibers and p-RMLC staining of transfected cells, an indication of an increase in phosphatase activity toward p-RMLC. These effects of MYPT3 occurred mainly at the cell membranes and cell edges, probably due to MYPT3 localization to the membrane through the CAAX tail. More obvious general effects on the loss of stress fibers were observed when the CAAX tail was removed, leading to an easily detectable phenotype. Interestingly, a mutant with nonphosphorylatable alanines (3A) was inactive, whereas acidic residue substitutions (3D) resulted in a constitutively active form, both in inducing morphological effects on stress fibers and in a myosin light chain phosphatase assay. Taken together, these results are consistent with the view that MYPT3 can be activated upon PKA phosphorylation.

Upon closer examination on the effects of phosphorylation on the three residues, we detected that PKA has a preference for Ser-353, followed by Ser-340, and the least activity to Ser-341 (Fig. 2E). However, we had observed that single serine to aspartate mutations are not sufficient to reduce AR/PS binding (data not shown), and thus it seems that although phosphorylation of Ser-353 is strongest, there appears to be an additional requirement for the phosphorylation of another site, or sites, for the reduction of AR/PS binding. Nevertheless, our study indicates that multiple phosphorylations within the PS region are required for the perturbation of the PS with the AR of MYPT3 and activation of the associated PP1c.

View larger version (77K): [in this window] [in a new window]   FIGURE 5. Myosin phosphatase activity with wild type and PS mutants. A, typical in vitro phosphatase assay results. Cells were co-transfected with either FLAG-FL, FL 3A, FL 3D, or PS mutants together with the HA-PP1c construct in COS7 cells. The complexes were immunoprecipitated and normalized for the HA-PP1c levels before subjecting to myosin phosphatase assays. Chart represents the statistical analysis of three independent assays with bars representing standard deviation. The Western blots represent the typical protein levels for the assays. B, HeLa cells transfected with the various constructs expressing FLAG-tagged proteins and HA-PP1c were treated with Me2SO (–Forskolin) or 40 µM forskolin (+Forskolin) before fixation in paraformaldehyde. Cells were co-stained with anti-FLAG and anti-phospho-RMLC antibody. Transfected cells are marked with asterisks. C, statistical analysis of reduction of staining in HeLa cells expressing wild type and mutant CAAX constructs. Bar chart denotes percentage of transfected cells showing a loss in filamentous p-RMLC staining in comparison to neighboring untransfected cells. Shaded bars are for transfected cells that were forskolin-treated. All statistics were generated from three independent experiments with counts of 100 transfected cells, and bars represent the standard deviation. Significant difference after forskolin treatment of p < 0.001 was indicated by asterisks.

View larger version (59K): [in this window] [in a new window]   FIGURE 6. Interactions of PS motif with N-terminal AR region in the regulation of myosin light chain phosphatase activity. A, detection of an interaction between PS and the AR regions. FLAG-AR was co-expressed with GST, GST-PS, or GST-PS mutants in COS7 cells, and the GST fusion proteins were affinity-purified in IP buffer. One set of GST-PS was phosphorylated with PKA as indicated. Bound proteins after extensive washing were separated on SDS-PAGE, and the relative amount of co-precipitated FLAG-AR was measured by Western blotting. B, PS inhibition of AR/PP1c myosin light chain phosphatase activity. Purified FLAG-AR/HA-PP1c complexes were preincubated with 2µg of PS proteins before being subjected to myosin light chain phosphatase assay. Chart is representative of three independent assays with error bars signifying S.D. The Western blots represent the typical protein levels in the assays. C, inhibitory effect of PS fragments on AR/PP1c-induced stress fiber loss. HeLa cells were co-transfected with HA-AR, HA-PP1c, and either FLAG-PS, FLAG-PS 3A, or FLAG-PS 3D constructs. Cells were fixed and stained with phalloidin for stress fibers. a and b, AR; c and d, AR + PS; e and f, AR + PS 3A; g and h, AR + PS 3D. Transfected cells are marked with asterisks.

View larger version (31K): [in this window] [in a new window]   FIGURE 7. Proposed model of MYPT3 activation. The C-terminal CAAX motif is usually prenylated and targets MYPT3 to the plasma membrane. In the nonphosphorylated state, the conserved phosphorylation site (PS) binds tightly to the N-terminal ankyrin repeat region and interferes with PP1c and/or RMLC recognition and hence myosin phosphatase activity. When PS is phosphorylated by PKA, it is released from the AR region, and myosin phosphatase activity is recovered.

We propose that an intramolecular or intermolecular interaction and a subsequent conformation change may play an essential role in the regulation of MYPT3 by PKA, because deletion, PKA phosphorylation, or aspartate mutations in the PS in MYPT3 all led to PP1c activation. With MYPT1, recent structural analysis has revealed that the complex formation of its N-terminal and ankyrin repeats with PP1c was accompanied by changing the shape and charges around the catalytic cleft of PP1c, which may determine substrate entry and specificity (14). This conformational change may not occur if the ankyrin repeat region is perturbed, thus resulting in PP1c being unable to interact with the substrate. Accordingly, we have observed a specific interaction of the nonphosphorylated conserved PS with the N-terminal ankyrin repeat region of MYPT3. This interaction was much reduced when the acidic mutants or the PKA phosphorylated PS was used, thus providing strong evidence that phosphorylation down-regulates this interaction. Furthermore, stress fiber losses induced by the AR could be inhibited by co-expression with the PS WT or PS 3A but not with the PS 3D mutant. Thus, in contrast to the other well studied members of the MYPT family, we conclude that MYPT3 is natively inactive and is activated rather than inactivated by phosphorylation, as has been demonstrated by other well studied MYPT members to date.

As MYPT1 has been shown to be phosphorylation targets for a variety of protein kinases (12, 13, 15, 17, 18), it remains to be seen if MYPT3 can also be phosphorylated by kinases other than PKA. On the other hand, PKA has been reported to interfere with actomyosin cytoskeleton rearrangements through a number of signaling pathways (37, 38). Phosphorylation of C-terminal Ser-188 of the small GTPase RhoA was found to affect its association with GDP dissociation inhibitor and ROK signaling events (33, 39). PKA is also known to directly phosphorylate and inhibit myosin light chain kinase (40, 41) and cause RMLC dephosphorylation indirectly. Phosphorylation by PKA of AKAP-Lbc, a Rho family exchange factor, and the subsequent recruitment of 14-3-3 has also been shown to down-regulate Rho exchange activity of AKAP-Lbc with subsequent Rho inhibition (42). More recently, PKA (and protein kinase G) was also shown to phosphorylate Ser-695 of MYPT1, which prevents the phosphorylation of the adjacent and inhibitory Thr-696 (20). Earlier work on phosphorylation of MYPT1 by PKA also affected its association with phospholipids and membrane association (26). Our finding is that PKA-dependent activation of MYPT3, presumably mostly at the cell membrane, indicates a widening role for PKA in regulating muscle contraction, endothelial cell barrier, and cell morphology.

	FOOTNOTES

 
^* This work was supported by GlaxoSmithKline (Singapore). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: GSK-IMCB Group, Institute of Molecular and Cell Biology, 61 Biopolis Dr., Singapore 138673/Dept. of Anatomy, National University of Singapore, Singapore. Tel.: 65-6586-9556; Fax: 65-6774-0742; E-mail: mcbthoml{at}imcb.a-star.edu.sg.

² The abbreviations used are: RMLC, regulatory light chain of myosin; MYPT3, myosin phosphatase targeting subunit 3; TIMAP, transforming growth factor--inhibited membrane-associated protein; PP1c, protein phosphatase 1; PKA, protein kinase A; MLCP, myosin light chain kinase and phosphatase; ROK, Rho kinase; AA, aliphatic amino acid; AR, ankyrin repeats; TRITC, tetramethylrhodamine isothiocyanate; HA, hemagglutinin; ATPS, adenosine 5'-O-(thiotriphosphate); GST, glutathione S-transferase; MRCK, myotonic dystrophy-related CDC42-binding kinase; PS, phosphorylation site.

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