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• W2010065280 abstract "The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCKα-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an α-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCKα-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1δ (PP1δ), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1δ, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the α-helical leucine repeats. The former was able to interact with PP1δ only in the phosphorylated state and result in inactivation of PP1δ activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.AF312028 The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCKα-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an α-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCKα-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1δ (PP1δ), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1δ, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the α-helical leucine repeats. The former was able to interact with PP1δ only in the phosphorylated state and result in inactivation of PP1δ activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.AF312028 myotonic dystrophy kinase-related Cdc42 binding kinase RhoA binding kinase myosin light chain 2 myosin binding subunit protein phosphatase 1 phosphorylation inhibitory motif α-helical leucine zipper catalytic domain polymerase chain reaction hemagglutinin polyvinylidene difluoride glutathione S-transferase adenosine 5′-O-(thiotriphosphate) expressed sequence tag The Rho subfamily of GTPases are biological regulators of actin cytoskeleton. In adherent cells, RhoA induces stress fiber formation, Rac-1 generates lamellipodia, and Cdc42 produces filopodia and actin microspikes (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5200) Google Scholar). A variety of effectors of these cytoskeletal switches has been isolated and characterized (see Refs. 2Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1666) Google Scholar and 3Lim L. Manser E. Leung T. Hall C. Eur. J. Biochem. 1996; 242: 171-185Crossref PubMed Scopus (273) Google Scholar for reviews), some of which are directly involved in regulation of actin dynamics. We and others have reported serine/threonine kinases related to the myotonic dystrophy kinase that play effector roles for the perspective GTPase in cytoskeletal reorganization (4Leung T. Chen X.-Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 5Matsui T. Amano M. Yamanoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (935) Google Scholar, 6Ishizaki T. Naiti M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (454) Google Scholar, 7Leung T. Chen X.-Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (220) Google Scholar). ROKs or Rho kinases are downstream effectors of RhoA in organizing stress fibers (4Leung T. Chen X.-Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar, 5Matsui T. Amano M. Yamanoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (935) Google Scholar, 6Ishizaki T. Naiti M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (454) Google Scholar), whereas MRCKs1 play an important role in Cdc42 functions in regulating actinomyosin dynamics in cultured cells (7Leung T. Chen X.-Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (220) Google Scholar). Precisely how these occur is not clear, although a number of proteins are known to be effective substrates for these kinases. These include the non-muscle myosin light chain 2 (MLC2), whose phosphorylation state is crucial for actinomyosin contractility and polymerization (7Leung T. Chen X.-Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (220) Google Scholar, 8Kureishi Y. Kobayashi S. Amano M. Kimura K. Kanaide H. Nakano T. Kaibuchi K. Ito M. J. Biol. Chem. 1997; 272: 12257-12260Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar), the myosin binding subunit p130 (9Amano M.K. Chihara K. Fukuta N. Nakamura Y. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (947) Google Scholar, 10Kawano Y. Fukata Y. Oshiro N. Amano M. Nakamura T. Ito M. Matsumura F. Inagaki M. Kaibuchi K. J. Cell Biol. 1999; 147: 1023-1038Crossref PubMed Scopus (473) Google Scholar, 11Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar), Ezrin, Radixin, and Moesin (ERM) family proteins (12Matsui T. Maeda M. Doi Y. Yonemura S. Amano M. Kaibuchi K. Tsukita S. Tsukita S. J. Cell Biol. 1998; 140: 647-657Crossref PubMed Scopus (725) Google Scholar), adducin (13Kimura K. Fukata Y. Matsuoka Y. Bennett V. Matsuura Y. Okawa K. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1998; 273: 5542-5548Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), and intermediate filament proteins (14Kosako H. Goto H. Yanagida M. Matsuzawa K. Fujita M. Tomono Y. Okigaki T. Odai H. Kaibuchi K. Inagaki M. Oncogene. 1999; 18: 2783-2788Crossref PubMed Scopus (105) Google Scholar, 15Sin W-C. Chen X-Q. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 6325-6339Crossref PubMed Google Scholar, 16Goto H. Kosako H. Tanabe K. Yanagida M. Sakurai M. Amano M. Kaibuchi K. Inagaki M. J. Biol. Chem. 1998; 273: 11728-11736Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 17Inada H. Togashi H. Nakamura Y. Kaibuchi K. Nagata K. Inagaki M. J. Biol. Chem. 1999; 274: 34932-34939Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), which are directly or indirectly linked to the actin cytoskeleton. In particular, the myosin binding subunit MBS130 appears to play a unique role in the regulation of the activity of the associated PP1 catalytic subunit. The specific binding of MBS130 to RhoA may link this regulatory subunit to Rho-dependent events (9Amano M.K. Chihara K. Fukuta N. Nakamura Y. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (947) Google Scholar). Indeed chicken gizzard MBS130 is found to be effectively and specifically phosphorylated by ROK at threonine 695 and serine 854 (10Kawano Y. Fukata Y. Oshiro N. Amano M. Nakamura T. Ito M. Matsumura F. Inagaki M. Kaibuchi K. J. Cell Biol. 1999; 147: 1023-1038Crossref PubMed Scopus (473) Google Scholar, 11Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). Phosphorylation at threonine 695 resulted in inhibition of the intrinsic phosphatase activity. Other proteins that can interact with and phosphorylate MBS130 include the cGMP-dependent protein kinase 1α and an unidentified mitotic kinase, but in contrast, such phosphorylation resulted in activation of protein phosphatase activity (18Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (438) Google Scholar, 19Totsukawa G. Yamakita Y. Yamashiro S. Hosoya H. Hartshorne D.J. Matsumura F. J. Cell Biol. 1999; 144: 735-744Crossref PubMed Scopus (54) Google Scholar). MBS130 therefore appears to serve as a scaffold for multiple protein interactions as well as phosphorylation regulation. Indeed, a recent report has indicated that a number of proteins involve in the Rho signaling pathways including Ezrin, Radixin and Moesin family proteins, adducin, and MBS130 can be found to colocalize at cell periphery upon stimulation. It is possible that MBS130 may provide a bridge for various Rho-dependent components to function in a coordinated manner (20Fukata Y. Kimura K. Oshiro N. Saya H. Matsuura Y. Kaibuchi K. J. Cell Biol. 1998; 141: 409-418Crossref PubMed Scopus (181) Google Scholar). Since the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) also consist of similar catalytic domains with differential cellular localization and cellular functions (7Leung T. Chen X.-Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (220) Google Scholar, 21Chen X-Q. Tan I. Leung T. Lim L. J. Biol. Chem. 1999; 274: 19901-19905Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), it is of interest to investigate if these kinases could share similar and different sets of substrates for their cellular activities. As a first step toward identification of new substrates for kinases such as MRCKs, we derived a filter assay to screen for potential candidates. This assay allows renatured proteins on the filter to be phosphorylated by specific recombinant kinases. Using this assay for MRCKα kinase, we identified two potential substrate proteins, one of which was MBS130; the other was a novel related protein. Full-length p85 cDNA was obtained by first performing a PCR reaction of the presumed initiation codon from the first exon of the human genomic DNA using two adaptor primers, 5′-CAGGATCCATGTCCGGAGAGGATGGC-3′ and 5′-GCAGGCCTGGTGCAGG-3′. This was then joined to aStuI/NotI fragment from EST clone AI1825921 that contains the rest of the 3′ end of p85. p85NT was derived from a BamHI/PstI DNA fragment of p85 (encoding residues 1–389), and p85CT was obtained by subcloning a PCR fragment from a 5′ primer 5′-CAGGATCCTGCCGCCTGCTGGCCG-3′ (encompassing residues 346–782) into pXJ-40 FLAG-tagged vector as previously described (4Leung T. Chen X.-Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar). A shorter C-terminal construct encompassing the conserved α-helix leucine zipper (p85αLZ; residues 591–782) was derived from PCR product using 5′ primer 5′-CGGATCCCGAAGGCCCCGCGTC-3′. In-frame deletions by Nar1 restriction enzyme digest, which removed a central conserved region (p85ΔNar; deleted residues 521–610), and by BssHII (p85ΔAR1), which deleted an ankyrin repeat (residues 51–71), were likewise subcloned. Mutagenesis was carried out as previously described (4Leung T. Chen X.-Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar) using primer pairs 5′-CTGCATGCAGCGGCACACTGG-3′ and 5′-CTGCATGCAGGGCAGAGGCGCC-3′ to generate p85ΔAR2 (deleted residues 232–264) and primer pairs 5′-GACGTCGACGGGCCGCACAGGGTGTGACTCTTAC3-′ and 5′-GACGTCGACTCTGGCGCATGAGACGG-3′ to generate p85S559A/T560A (p85AA). PP1δ was derived from EST clone AI156840, which contains the whole of the coding sequence. The nucleotide sequences encoding the 50 amino acids ofphosphorylation inhibitory motif (PIM; residues 526–575) of the wild-type p85 and mutant p85AA were obtained by PCR using primer pairs 5′-CAGGATCCCGGGACCGACGGAG-3′ and pXJ40 reverse primer. The 156-base pair BamHI/PstI-digested PCR fragments were subcloned into pXJ40-GST vector for mammalian cell transfection (22Zhao Z-S. Manser E. Chen X-Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar) and into pGEX-4T2 for the production of GST-PIM50 and GST-PIM50AA fusion proteins. The N terminus containing the kinase domain of MRCKα (residues 1–473) was obtained by subcloning a 1.4-kilobase BamHI/HindIII fragment from full-length MRCKα into pGEX4T2 to generate a vector producing MRCKα-CAT. GST-MLC2 was prepared as previously described (7Leung T. Chen X.-Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (220) Google Scholar). COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10‥ fetal bovine serum, and HeLa cells were cultivated in modified Eagle's medium with 10‥ fetal bovine serum (Hyclone). Subconfluent HeLa cells plated on coverslips for 48 h were transfected with various HA- or FLAG-tagged DNA constructs (1 μg/ml) with LipofectAMINE (Life Technologies, Inc.) according to recommended protocol. 16 h after transfection, cells were fixed with 4‥ paraformaldehyde and stained with the combination of various primary antibodies: anti-HA (12CA5; Roche Molecular Biochemicals) or anti-FLAG (M2; Sigma). Stained cells were analyzed with an MRC 600 confocal imager adapted to a ZEISS Axioplan microscope. For localization studies, transfected HeLa cells were serum-starved for 4–6 h before treatment with lysophosphatidic acid (300 ng/ml; Sigma) or phorbol myristic acetate (100 ng/ml; Sigma). COS-7 cells grown in Dulbecco's modified Eagle's medium with 10‥ fetal bovine serum were similarly transfected with various constructs. 24 h after transfection, cell extracts were obtained with lysis buffer ((25 mm HEPES, pH 7.7, 0.15m NaCl, 1.5 mm MgCl2,0.2 mm EDTA, 1 mm sodium vanadate, 20 mm β-glycerol phosphate, 5‥ glycerol, 0.1‥ Triton X-100 and 1× inhibitor mix (Roche Molecular Biochemicals)), separated on a SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with anti-HA or anti-FLAG antibodies for expression. Tissue extracts from rat brain and other tissues or cultured cells were resolved on either one- or two-dimensional gels, electro-transferred onto PVDF filters, and renatured in buffer containing phosphate-buffer saline with 0.5 mm MgCl2, 1 mm dithiothreitol, 1‥ bovine serum albumin, and 0.1‥ Triton X-100 for 2 h. Filters were washed with phosphate-buffered saline, 0.1‥ Triton, incubated with a phosphorylation mixture containing 20 mm Tris-HCl, 75 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 5 μg/ml GST-MRCKα-CAT fusion protein, and 10 μCi/ml [γ-32P]ATP for 30 min, and washed with phosphate-buffered saline, 0.1‥ Triton and 6 mguanidinium chloride before autoradiography. Control experiments were also performed using GST-α-p21-activated kinase (22Zhao Z-S. Manser E. Chen X-Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar) and protein kinase A (Sigma). Kinase assays were carried out in kinase buffer containing 20 mm Tris-HCl pH 7.5, 75 mm NaCl, and 10 mm MgCl2 as previously described (5Matsui T. Amano M. Yamanoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (935) Google Scholar). For labeling of GST-MLC2, purified fusion protein (10 μg) on glutathione beads was subjected to phosphorylation using 20 μg/ml GST-MRCK-α-CAT and 1 μm[γ-33P]ATP for 1 h at 30 °C. After an extensive wash with GST purification buffer, the phosphorylated protein was eluted with 5 mm reduced glutathione. Phosphatase assays were carried out at 30 °C using 33P-GST-MLC2 as substrate. To show phophorylation-mediated PP1 inhibition, the immunoprecipitated FLAG-p85·HA-PP1δ complex was first incubated in 20 μl of kinase buffer with 0.15 mm ATPγS in the presence or absence of 0.5 μg of GST-MRCKα-CAT for 30 min at 30 °C. Phosphatase assays were initiated by the addition of 5 μm33P-GST-MLC2 in 30 mmTris-HCl, PH 7.5, 0.1 m KCl, 2 mmMgCl2, and 0. 1 mg/ml bovine serum albumin. The reactions were stopped by adding an equal volume of SDS sample buffer at each time point indicated and boiling for 5 min before gel loading. To show PP1 inhibition by phosphorylated GST-PIM50, 10 μg of the fusion protein was first incubated in kinase buffer (as above using ATPγS) with and without GST-MRCKα-CAT for 30 min at 30 °C. These phosphorylated and nonphosphorylated GST-PIM50 proteins were then separately mixed with the immunoprecipitated FLAG-p85·HA-PP1δ complex and preincubated for 1 min before the start of phosphatase assays. Phosphatase activities were quantified using the Molecular Dynamics PhosphorImager System. COS-7 cells co-expressing various FLAG-p85 and HA-PP1δ constructs were lysed in buffer containing 25 mm HEPES, pH 7.3, 0.15 mNaCl, 0.5 mm MgCl2, 0.2 mm EDTA, 20 mm β-glycerol phosphate, 1 mm sodium vanadate, 5‥ glycerol, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 0.1‥ Triton, 1 μg/ml each aprotinin, leupeptin, and pepstatin A, and 1× protease inhibitor mixture and incubated with anti-FLAG-conjugated agarose beads (Sigma) for 1 h at 4 °C. After an extensive wash, the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and subjected to immunoblot analysis using anti-FLAG or anti-HA antibodies as mentioned. To detect any interaction of these complex with the phosphorylated GST-PIM50, an in vitro assay containing either the unphosphorylated or phophorylated GST-PIM50 was added to COS-7 cell lysates co-expressing FLAG-p85AA and HA-PP1δ and followed by immunoprecipitation using anti-FLAG-conjugated agarose beads. Immunoblot analyses were carried out as before. Northern blots containing mRNA from various human tissues were obtained fromCLONTECH and were hybridized with a full-length32P-labeled p85 probe as previously described (5Matsui T. Amano M. Yamanoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (935) Google Scholar). One- and two-dimensional gel analyses were carried out according to standard protocol, and separated proteins were transferred to PVDF membranes (PerkinElmer Life Sciences) and probed with various antibodies, including an antibody to a phosphorylated threonine 695 of chicken gizzard M130 (which is identical to the flanking sequence around threonine 560 of p85). Analysis of tryptic peptides and phosphopeptides were performed with a QSTAR Mass-spectrometer (PerkinElmer). To identify and characterize potential substrates for ROKα and MRCKα, we made GST fusion proteins of the catalytic domains of both ROKα (1) and MRCKα (1). The yield and catalytic activities of MRCKα-CAT were consistently higher than ROKα-CAT, and subsequent experiments were thus carried out with GST-MRCKα-CAT. Here we observed that renatured proteins separated on SDS-polyacrylamide electrophoresis gels and transferred onto PVDF membrane filters were readily phosphorylated by MRCKα-CAT. Two proteins of 130 and 85 kDa were prominently and specifically phosphorylated by MRCKα-CAT (Fig. 1) but not by α-p21-activated kinase or protein kinase A (data not shown). These two proteins are not abundant in tissues such as brain and testis but could easily be enriched by passing through an affinity dye Reactive Brown 10-Sepharose column (Sigma), and this constitutes a simple one-step enrichment of these proteins for further purification (Fig. 1 A). Further separation of these two proteins was achieved with two-dimensional gel electrophoresis (Fig.1 B), and the Coomassie Blue-stained spots corresponding to the phosphorylated proteins were excised for peptide sequencing. Both peptide sequencing and subsequent immunoblotting analyses showed that the p130 corresponds to the previously reported MBS130 (data not shown). Peptide sequence analysis also indicated that the p85 is a novel protein (Fig.2 A). A 2.8-kilobase human EST clone AI825921 contains most of the coding sequence except for the extreme N terminus. The complete match of this cDNA with two overlapping genomic clones, S51329 and AC005782, from human chromosome 19 (Fig. 2 C) suggested that the N terminus is confined within the first exon. A full-length cDNA for p85 was thus constructed from the PCR product of the first exon of human p85 genomic DNA and subsequently joined to the truncated cDNA. The amino acid sequence derived from this cDNA indicated that p85 is structurally related to MBS130 (Fig. 2 B). The N terminus of p85 consists of a closely related structure with 6 ankyrin repeats and 48‥ identity to MBS130, which has been reported to have 7–8 repeats (23Chen Y.H. Chen M.X. Alessi D.R. Campbell D.G. Shanahan C. Cohen P. Cohen P.T. FEBS Lett. 1994; 356: 51-55Crossref PubMed Scopus (128) Google Scholar, 24Shimizu H. Ito M. Miyahara M. Ichikawa K. Okubo S. Konishi T. Naka M. Tanaka T. Hirano K. Hartshorne D.J. J. Biol. Chem. 1994; 269: 30407-30411Abstract Full Text PDF PubMed Google Scholar). A putative PP1 binding consensus sequence, RTVRF (25Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (531) Google Scholar), was also present immediately before the ankyrin repeats. The C terminus contains a conserved α-helical structure with leucine zippers at the C-terminal end (αLZ). This structure can form homodimers or heterodimers (e.g. with M20, which also contains this structural motif (26Johnson D. Cohen P. Chen M.X. Chen Y.H. Cohen P.T. Eur. J. Biochem. 1997; 244: 931-939Crossref PubMed Scopus (83) Google Scholar)). Of most striking similarity (87‥ identity) is a central motif, which contains the sole phosphorylation site for MRCKα-CAT (refer to Fig. 3).Figure 3Mapping of phosphorylation sites of p85 by MRCK α-CAT. A, a schematic diagram of the various p85 constructs. AR, ankyrin repeats; αLZ; PIM; mutations in PIM50AA (S559A/T560A) were indicated by ××. B, immunoprecipitated wild-type FLAG-tagged p85 (lane 1) and the various truncated mutants (p85ΔNar1 (lane 2), p85CT (lane 3), p85CTΔNar1 (lane 4), p85NT (lane 5), and p85αLZ (lane 6)) and 5 μg of GST-PIM50 (lane 7) and mutant PIM50AA(lane 8) were phosphorylated with MRCKα-CAT and [32P]ATP. Proteins were resolved on a 10‥ polyacrylamide gel electrophoresis-SDS gel for Coomassie Blue staining (top) and autoradiography (bottom).Arrowheads indicate the positions of IgG light and heavy chains; the position for the kinase autophosphorylation was marked byarrows. C, FLAG-tagged p85 was expressed alone or together with either HA-tagged MRCKα-CAT or ROKα-CAT, and the proteins transferred to PVDF filters were detected separately with anti-FLAG, anti-p85-pT, and anti-HA to detect expression of the kinases. FLAG-tagged immunoprecipitated p85 before and after phosphorylation in vitro by GST-MRCKα-CAT was also shown as controls for comparison (right panel). WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The genomic organization of p85 showed that the mRNA is derived from 22 exons (Fig. 2 C). Intriguingly, a number of integration hotspots (AAVS1) of adenovirus-associated virus (AAV) was found in the first exon/intron regions of p85 genomic sequence (Fig.2 D; Refs. 27Kotin R.M. Linden R.M. Berns K.I. EMBO J. 1992; 11: 5071-5078Crossref PubMed Scopus (402) Google Scholar and 28Recchia A. Parks R.J. Lamartina S. Toniatti C. Pieroni L. Palombo F. Ciliberto G. Graham F.L. Cortese R. La Monica N. Colloca S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2615-2620Crossref PubMed Scopus (156) Google Scholar). The consequence of these integrations is not known but is expected to disrupt the expression of this gene. Rearrangements and disruption of a nearby troponin gene were also observed upon adenovirus-associated virus integration (29Dutheil N. Shi F. Dupressoir T. Linden R.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4862-4866Crossref PubMed Scopus (81) Google Scholar). Northern blot analysis indicated that the 3-kilobase p85 mRNA was ubiquitously expressed and is especially high in the heart (Fig.2 E). P85 protein expressed in serum-starved HeLa cells mainly showed cytoplasmic punctate distribution (Fig. 2 F, a) but was readily redistributed to cell peripheral upon treatment with lysophosphatidic acid and phorbol myristic acetate (Fig. 2 F, b and c). To confirm the nature of phosphorylation of p85, we expressed the FLAG-tagged wild-type and deletion mutants of p85 in COS-7 cells (Fig. 3 A). The immunoprecipitated proteins were phosphorylated with MRCKα-CAT to map the phosphorylation site(s). It is clear that the major site(s) is within the central conserved region, as mutants deleted of this region were not phosphorylated (Fig. 3 B, lanes 2 and4). To confirm this, we expressed GST fusion protein containing the wild-type conserved PIM motif (PIM50; also refer to Fig.4) and a mutant S559A/T560A (PIM50AA) and showed that the mutant was not phosphorylated (Fig. 3 B, lane 8), unlike the wild-type control (lane 7). This in vitro phosphorylation site of p85 on threonine 560 by MRCKα-CAT was further confirmed by mass spectrometry on the tryptic phosphopeptides derived from p85 phosphorylation. Further evidence was obtained by probing p85 protein expressed from COS-7 cells that were co-transfected with either MRCKα-CAT or ROKα-CAT with an antibody that specifically recognizes phosphorylated threonine 560 of p85. Clearly, threonine 560 of p85 can be phosphorylated by both MRCKα-CAT and ROKα-CAT in vivo(Fig. 3 C). To see if p85 can associate with PP1, we co-transfected FLAG-tagged p85 and HA-tagged PP1α, -γ, and -δ isoforms. Only PP1δ isoform was immunoprecipitated with p85, indicating a specific interaction between p85 and PP1δ. This was also evident from the co- immunoprecipitation of the endogenous PP1δ with the overexpressed p85 (data not shown). Constructs with an intact N terminus, including the ankyrin repeats, could effectively interact with PP1δ (Fig.4 A, lanes 1–4), whereas a deletion mutant of a single ankyrin repeat (lane 5) can dramatically reduce such interaction. Deletion mutants devoid of N terminus are totally ineffective (Fig. 4 A, lanes 6 and7). The N terminus of p85 could also interact with MLC2 (Fig.4 B), and the C terminus is totally ineffective. Hence PP1δ can form a tight complex with p85 and substrate MLC2 through its N terminus. Next, to examine if the phosphorylation of threonine 560 by MRCKα-CAT can regulate PP1 activity, we measured the time course of dephosphorylation toward 33P-MLC2. As shown in Fig.4 C, nonphosphorylated wild-type p85 (p85WT) or the phosphorylation-defective p85 mutant (p85AA) were equally active in MLC2 dephosphorylation. Wild-type p85 but not the mutant p85AA, when phosphorylated in vitro with MRCKα-CAT, showed significant reduction in the rate of MLC2 dephosphorylation. These results confirm a similar observation with MBS130 where phosphorylation of a conserved threonine 695 within a highly conserved motif was essential for the inhibition of phosphatase catalytic activity (11Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). Based on the biochemical and functional similarities between p85 and MBS130, we therefore designate p85 as MBS85. Because PP1 activities are essential for regulating the phosphorylation states of myosin, it is likely that the biochemical interaction of MBS85 with PP1δ may be correlated with morphological effects in cultured cells. Furthermore adherent cells mainly exhibit active Rho phenotype in serum medium, and the interference of endogenous PP1δ would be expected to affect this actin structure. As shown in Fig.5 A, expression of wild-type p85 alone did not give any obvious morphological consequence. However, truncation of the C terminus led to various degrees of disassembly of actin stress fibers, with the most striking effects when both the PIM and αLZ motifs were totally removed (p85NT; Fig. 5,A and B). Deletion of the PIM motif (p85ΔNar1) or mutation of the phosphorylation site (p85AA) was significantly effective in inducing similar morphological effects, although this appears to depend on the levels of expression (Fig. 5 A, bottom panel). Both the PIM and αLZ motifs at the C terminus appear to exert opposing effects on the N-terminal function. Truncation of the N terminus resulted in general increases in actin polymerization (p85CT in Fig. 5 A) that was resistant to C3 treatment (data not shown). As αLZ alone was also effective, although to a lesser extent (data not shown), it is likely that PIM and αLZ domains have independent activities toward actin assembly. Similar trends were also observed when the various constructs were co-expressed with PP1δ (Fig. 5 B). In this case, the morphological effects were more pronounced in the presence of exogenous PP1δ. As expected, both ankyrin repeat mutants p85ΔAR1and p85ΔAR2 that were defective in binding to PP1δ were totally ineffective in inducing morphological changes (Fig.5 B and data not shown). Hence the phenotypic effects of the various MBS85 variants on actin cytoskeleton correlate well with the biochemical data described earlier. It is known that phosphorylation of threonine 695 of MBS (which is equivalent to the threonine 560 of p85) is critical for the inhibitory effects on PP1 catalytic function (11Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). It is likely that a similar mechanism may operate for MBS85. To test this possibility, we derived an assay to examine the effect of phosphorylation on the interaction of the highly conserved GST-PIM with p85/PP1δ complex. The phosphorylation-deficient mutant p85AA was used to eliminate possible competition for binding. As shown in Fig. 6 A, phosphorylated GST-PIM, but not the nonphosphorylated form, was detected in the p85/PP1δ immuno-complex. Similarly only glutathione beads with the phosphorylated GST-PIM, but not the nonphosphorylated or phosphorylation-deficient mutant GST-PIM50AA, were able to pull down the expressed PP1δ or p85/PP1δ complex (Fig.6 B), clearly indicating that phosphorylation of threonine 560 of MBS85 is essential for its interaction with PP1δ. To examine if such interaction is functional, we measured the catalytic activities of the immuno-complex in the presence of in vitrophosphorylated or nonphosphorylated GST-PIM50. Phosphorylated GST-PIM50, but not the nonphosphorylated form, was likewise effective in inhibiting the catalytic activity of p85·PP1δ complex (Fig.6 C). This provides further evidence that the interaction is functional. Next we tested the effects of co-expression of this PIM50 motif on the p85NT-induced morphological changes. As shown in Fig.7, A and B, PIM50 expression is sufficient in reversing the effect of p85NT-induced actin stress fiber losses. The phosphorylation-deficient mutant PIM50AA was totally inefficient (Fig. 7 B). These inhibitory effects became less pronounced when p85NT was co-expressed with PP1δ, suggesting that PIM50 may well be competing with the catalytic subunit in regulating actin dynamics. We therefore conclude that from both biochemical and morphological data that the central conserved motif of MBS85 can be regulated by phosphorylation, resulting in conformational changes that affect the associated PP1δ catalytic property and subsequently effects on actin morphology, probably through the eventual effects on myosin phosphorylation. Two candidate proteins from rat brain cytosolic extract were identified in this study on the basis of their in vitrophosphorylation by MRCKα-CAT. The p130 protein was confirmed to be MBS130 by peptide sequencing and immunoreactive toward specific antibodies. The smaller protein p85 is a novel protein that is structurally related to MBS130. Overall p85 shares low similarity to MBS130 (<40‥). The N terminus of p85 contains six ankyrin repeats that are known to be involved in protein-protein interactions. This motif shares a 48‥ identity to MBS130, which has 7–8 repeats (23Chen Y.H. Chen M.X. Alessi D.R. Campbell D.G. Shanahan C. Cohen P. Cohen P.T. FEBS Lett. 1994; 356: 51-55Crossref PubMed Scopus (128) Google Scholar,24Shimizu H. Ito M. Miyahara M. Ichikawa K. Okubo S. Konishi T. Naka M. Tanaka T. Hirano K. Hartshorne D.J. J. Biol. Chem. 1994; 269: 30407-30411Abstract Full Text PDF PubMed Google Scholar). Preceding these repeats is a short stretch (RTVRF) that resembles PP1 binding consensus sequence (25Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (531) Google Scholar). The C terminus of p85 is also conserved and consists of an α-helical structure with four leucine heptad repeats at the C-terminal end. This motif is known to be involved in dimerization and interaction with other proteins. M20, a small subunit protein of PP1, is found to be an integral part of the heterotrimeric complex (28Recchia A. Parks R.J. Lamartina S. Toniatti C. Pieroni L. Palombo F. Ciliberto G. Graham F.L. Cortese R. La Monica N. Colloca S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2615-2620Crossref PubMed Scopus (156) Google Scholar), and it contains a similar helical structure. Indeed, it has been reported that this M20 may be a spliced product from skeletal muscle isoform of MBS130 (30Hartshorne D.J. Hirano K. Mol. Cell Biochem. 1999; 90: 79-84Crossref Google Scholar). More recently, it has also been reported that other proteins can interact with the C terminus of MBS130. This includes RhoA and cGMP-dependent protein kinase 1α (9Amano M.K. Chihara K. Fukuta N. Nakamura Y. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (947) Google Scholar, 18Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (438) Google Scholar). The former links the PP1 complex to Rho-dependent regulatory events, and the kinase phosphorylates and activates PP1 activity. The presence of a similar motif in p85 at the C terminus suggests that it may serve similar functional roles. Indeed p85 can readily translocate to peripheral membrane upon treatment with lysophosphatidic acid and phorbol ester, factors that are known to have prominent effects on Rho GTPases and actin cytoskeleton (2Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1666) Google Scholar). A central conserved motif that contains the sole phosphorylation site for MRCKα and ROKα exhibits a more striking similarity. Threonine 560 of p85 is equivalent to threonine 695 of MBS130 and is located within a ∼50-amino acid conservative phosphorylation inhibitory motif (PIM50). Phosphorylation of the threonine 695 by ROKα has been shown to inhibit associated PP1 activity (11Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 30Hartshorne D.J. Hirano K. Mol. Cell Biochem. 1999; 90: 79-84Crossref Google Scholar). A second phosphorylation site (serine 854) by ROKα has also been described in MBS130, but this is absent in p85 (Fig. 2 A and Ref. 10Kawano Y. Fukata Y. Oshiro N. Amano M. Nakamura T. Ito M. Matsumura F. Inagaki M. Kaibuchi K. J. Cell Biol. 1999; 147: 1023-1038Crossref PubMed Scopus (473) Google Scholar). Here we have also demonstrated that p85 is functionally similar to MBS130. First it is specifically associated with PP1δ, and this depends on the N terminus including the whole of the ankyrin repeats. N-terminal as well as ankyrin-repeat deletion mutants are ineffective in binding PP1δ. In agreement with a previous report for MBS130 (32Ichikawa K. Hirano K. Ito M. Tanaka J. Nakano T. Hartshorne D.J. Biochemistry. 1996; 35: 6313-6320Crossref PubMed Scopus (77) Google Scholar), we also detected binding of the substrate MLC2 within the N-terminal region. In this respect, the N terminus of p85 alone can therefore act as scaffold for PP1 and myosin and confers specific phosphatase activity on MLC2 substrate (30Hartshorne D.J. Hirano K. Mol. Cell Biochem. 1999; 90: 79-84Crossref Google Scholar). p85 (designated here as MBS85) is therefore a genuine myosin binding subunit of PP1δ, similar to MBS130. Furthermore, the phosphorylation of threonine 560 of the central motif of MBS85 resulted in inhibition of PP1δ activity, also suggesting a conservation of phosphorylation mechanism in regulating the catalytic event. Similar conclusions can be derived from the morphological assays that clearly reflect the biochemical interactions. HeLa cells expressing the N terminus of MBS85 alone exhibits the greatest loss in actin stress fibers, an indicator for activation of PP1 in vivo. Interestingly, the two conserved motifs (PIM and αLZ) appear to act independently to counteract this N-terminal function as deletion of either motif attenuates the stress fiber losses. Overexpression of C-terminal αLZ motif alone can induce actin stress fibers that are resistant to C3 treatment, suggesting that this activates an event downstream of Rho. The exact mechanism of how this αLZ motif works is currently not clear. One possibility is that this motif may directly compete with binding proteins such as cGMP-dependent kinase 1α, which is known to activate PP1 (18Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (438) Google Scholar). Furthermore, the αLZ motif is known to be able to form homodimers or heterodimers with the small subunit M20, although the biological functions of these complexes are currently not known (data not shown; Refs. 26Johnson D. Cohen P. Chen M.X. Chen Y.H. Cohen P.T. Eur. J. Biochem. 1997; 244: 931-939Crossref PubMed Scopus (83) Google Scholar and 30Hartshorne D.J. Hirano K. Mol. Cell Biochem. 1999; 90: 79-84Crossref Google Scholar). We conclude from both biochemical and morphological analyses that different domains of the MBS85 have opposing activities in regulating actin polymerization and that the phosphorylation of threonine 560 in a central motif appears to fine tune these events. It is therefore important to demonstrate a direct interaction of the conserved central motif (PIM50) with PP1δ. Such an interaction occurred only when the threonine 560 was phosphorylated. Moreover, not only did such a motif play a role in intramolecular interaction, it is also equally effective both in vitro and in vivoin promoting intermolecular interactions when introduced separately. This leads to the conclusion that phosphorylation of MBS85 by myotonic dystrophy kinase-related kinases such as MRCKα and ROKα induces a conformational change at the central conserved motif that results in higher affinity toward PP1δ. Such an interaction may change the orientation or catalytic properties of PP1δ toward the associated myosin (through MLC2; Fig. 6 C), resulting in myosin phosphorylation and subsequent cytoskeletal changes. This is depicted as a working model in Fig. 8. It remains unclear as to which GTPase(s) and downstream kinase(s) are regulating this event, as endogenous levels of p85 in cultured cells are much lower than p130MBS, and the specific antibody that recognizes the phosphorylated peptide was unable to detect the endogenous phosphoprotein. Further experiments are therefore required to clarify this issue. In summary, we have isolated a novel myosin binding subunit that is ubiquitously expressed. Compared with MBS130, the smaller size and simpler arrangement of the regulatory domains of this novel MBS85 allow an easier analysis of structure and function relationships. The identification of an increasing number of these myosin binding subunits, which share a similar regulatory mechanism, should help to understand how each of these are regulated by various diverse signaling pathways in the control of the actin cytoskeleton. We thank Dr. M. Ito for the antibody to phosphorylated threonine 695 of MBS130 and Dr. Robert Qi for peptide sequence analysis." @default.
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• W2010065280 title "Phosphorylation of a Novel Myosin Binding Subunit of Protein Phosphatase 1 Reveals a Conserved Mechanism in the Regulation of Actin Cytoskeleton" @default.
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