FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4313856862> ?p ?o ?g. }

• W4313856862 endingPage "102893" @default.
• W4313856862 startingPage "102893" @default.
• W4313856862 abstract "The subcellular localization, activity , and substrate specificity of the serine/threonine protein phosphatase 1 catalytic subunit (PP1cat) is mediated through its dynamic association with regulatory subunits in holoenzyme complexes. While some functional overlap is observed for the three human PP1cat isoforms, they also show distinct targeting based on relative preferences for specific regulatory subunits. A well-known example is the preferential association of MYPT1 with PP1β in the myosin phosphatase complex. In smooth muscle, MYPT1/PP1β counteracts the muscle contraction induced by phosphorylation of the light chains of myosin by the myosin light chain kinase. This phosphatase complex is also found in nonmuscle cells, where it is targeted to both myosin and nonmyosin substrates and contributes to regulation of the balance of cytoskeletal structure and motility during cell migration and division. Although it remains unclear how MYPT1/PP1β traffics between microtubule- and actin-associated substrates, our identification of the microtubule- and actin-binding protein SPECC1L in both the PP1β and MYPT1 interactomes suggests that it is the missing link. Our validation of their association using coimmunoprecipitation and proximity biotinylation assays, together with the strong overlap that we observed for the SPECC1L and MYPT1 interactomes, confirmed that they exist in a stable complex in the cell. We further showed that SPECC1L binds MYPT1 directly and that it can impact the balance of the distribution of the MYPT1/PP1β complex between the microtubule and filamentous actin networks. The subcellular localization, activity , and substrate specificity of the serine/threonine protein phosphatase 1 catalytic subunit (PP1cat) is mediated through its dynamic association with regulatory subunits in holoenzyme complexes. While some functional overlap is observed for the three human PP1cat isoforms, they also show distinct targeting based on relative preferences for specific regulatory subunits. A well-known example is the preferential association of MYPT1 with PP1β in the myosin phosphatase complex. In smooth muscle, MYPT1/PP1β counteracts the muscle contraction induced by phosphorylation of the light chains of myosin by the myosin light chain kinase. This phosphatase complex is also found in nonmuscle cells, where it is targeted to both myosin and nonmyosin substrates and contributes to regulation of the balance of cytoskeletal structure and motility during cell migration and division. Although it remains unclear how MYPT1/PP1β traffics between microtubule- and actin-associated substrates, our identification of the microtubule- and actin-binding protein SPECC1L in both the PP1β and MYPT1 interactomes suggests that it is the missing link. Our validation of their association using coimmunoprecipitation and proximity biotinylation assays, together with the strong overlap that we observed for the SPECC1L and MYPT1 interactomes, confirmed that they exist in a stable complex in the cell. We further showed that SPECC1L binds MYPT1 directly and that it can impact the balance of the distribution of the MYPT1/PP1β complex between the microtubule and filamentous actin networks. Reversible protein phosphorylation is the most common posttranslational modification, acting as a molecular switch that can modulate protein conformation and/or protein–protein interactions. This in turn leads to alterations in enzymatic activity, subcellular localization, turnover of targets or signaling by other post-translational modifications (PTMs). Phosphoregulation plays a role in most cellular processes, including signaling, metabolism, migration, and cell cycle progression and is a key therapeutic target in diseases in which these processes are dysregulated. The predominant phosphorylated amino acid is serine (Ser), which accounts for >80% of phosphorylation events (1Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. et al.Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2860) Google Scholar). Threonine (Thr) and tyrosine (Tyr) account for the bulk of the remaining phospho-sites, with phosphorylation also demonstrated to a lesser extent on other amino acid residues (2Venerando A. Cesaro L. Pinna L.A. From phosphoproteins to phosphoproteomes: A historical account.FEBS J. 2017; 284: 1936-1951Crossref PubMed Scopus (23) Google Scholar). Protein phosphatase 1 (PP1) catalytic subunit (PP1cat) is ubiquitously expressed in eukaryotic cells and estimated to account for up to 70% of Ser/Thr dephosphorylation events (3Roadcap D.W. Brush M.H. Shenolikar S. Identification of cellular protein phosphatase-1 regulators.Methods Mol. Biol. 2007; 365: 181-196PubMed Google Scholar). Mammalian PP1cat is found primarily as three isoforms (α, β/δ, γ) that are encoded by three distinct genes (4Korrodi-Gregório L. Esteves S.L.C. Fardilha M. Protein phosphatase 1 catalytic isoforms: Specificity toward interacting proteins.Transl Res. 2014; 164: 366-391Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). These isoforms are >89% identical in amino acid sequence, with minor variations primarily at their NH2 and COOH termini (5Cohen P.T.W. Protein phosphatase 1--targeted in many directions.J. Cell. Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). Loss of function and biochemical studies of individual PP1 isoforms in eukaryotic organisms suggest some level of compensation or overlapping function, while also highlighting distinct phenotypes associated with the disruption of a single gene (4Korrodi-Gregório L. Esteves S.L.C. Fardilha M. Protein phosphatase 1 catalytic isoforms: Specificity toward interacting proteins.Transl Res. 2014; 164: 366-391Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In the cell, PP1 is regulated and achieves its substrate specificity through the association of the catalytic subunit with a range of regulatory or “targeting” subunits (6Heroes E. Lesage B. Görnemann J. Beullens M. Van Meervelt L. Bollen M. The PP1 binding code: A molecular-lego strategy that governs specificity.FEBS J. 2013; 280: 584-595Crossref PubMed Scopus (221) Google Scholar). This results in the combinatorial generation of a large and diverse group of dimeric PP1 holoenzyme complexes, each with its own subset of substrates and mechanism(s) of regulation. To date, >200 confirmed PP1-interacting proteins have been identified using a range of proteomic, bioinformatic, yeast two-hybrid, and biochemical approaches (7Trinkle-Mulcahy L. Andersen J. Lam Y.W. Moorhead G. Mann M. Lamond A.I. Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability.J. Cell Biol. 2006; 172: 679-692Crossref PubMed Scopus (210) Google Scholar, 8Moorhead G.B.G. Trinkle-Mulcahy L. Nimick M. De Wever V. Campbell D.G. Gourlay R. et al.Displacement affinity chromatography of protein phosphatase one (PP1) complexes.BMC Biochem. 2008; 9: 1-10Crossref PubMed Scopus (60) Google Scholar, 9Chamousset D. De Wever V. Moorhead G.B. Chen Y. Boisvert F.-M. Lamond A.I. et al.RRP1B targets PP1 to mammalian cell nucleoli and is associated with Pre-60S ribosomal subunits.Mol. Biol. Cell. 2010; 21: 4212-4226Crossref PubMed Scopus (35) Google Scholar, 10Bennett D. Lyulcheva E. Alphey L. Hawcroft G. Towards a comprehensive analysis of the protein phosphatase 1 interactome in Drosophila.J. Mol. Biol. 2006; 364: 196-212Crossref PubMed Scopus (22) Google Scholar, 11Fardilha M. Esteves S.L.C. Korrodi-Gregório L. Vintém A.P. Domingues S.C. Rebelo S. et al.Identification of the human testis protein phosphatase 1 interactome.Biochem. Pharmacol. 2011; 82: 1403-1415Crossref PubMed Scopus (60) Google Scholar, 12Flores-Delgado G. Liu C.W.Y. Sposto R. Berndt N. A limited screen for protein interactions reveals new roles for protein phosphatase 1 in cell cycle control and apoptosis.J. Proteome Res. 2007; 6: 1165-1175Crossref PubMed Scopus (45) Google Scholar, 13Yadav L. Tamene F. Göös H. van Drogen A. Katainen R. Aebersold R. et al.Systematic analysis of human protein phosphatase interactions and dynamics.Cell Syst. 2017; 4: 430-444.e5Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The majority of known PP1 regulatory proteins contain an RVxF docking motif that mediates association with a hydrophobic pocket in PP1cat (14Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1.EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (539) Google Scholar). Several contain additional PP1-binding sequences, such as SILK and MyPhoNE motifs, that enhance binding and contribute to isoform preference (see (6Heroes E. Lesage B. Görnemann J. Beullens M. Van Meervelt L. Bollen M. The PP1 binding code: A molecular-lego strategy that governs specificity.FEBS J. 2013; 280: 584-595Crossref PubMed Scopus (221) Google Scholar) for review). There is also a subset of inhibitory PP1 interactors that do not contain a typical RVxF docking motif (e.g., Inh2, SDS22) and can associate with PP1 bound to another regulatory subunit in trimeric complexes (6Heroes E. Lesage B. Görnemann J. Beullens M. Van Meervelt L. Bollen M. The PP1 binding code: A molecular-lego strategy that governs specificity.FEBS J. 2013; 280: 584-595Crossref PubMed Scopus (221) Google Scholar). Our interactome screens comparing the three human PP1 phosphatase isoforms have identified and characterized numerous novel proteins not previously defined as phosphatase complex members (7Trinkle-Mulcahy L. Andersen J. Lam Y.W. Moorhead G. Mann M. Lamond A.I. Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability.J. Cell Biol. 2006; 172: 679-692Crossref PubMed Scopus (210) Google Scholar, 9Chamousset D. De Wever V. Moorhead G.B. Chen Y. Boisvert F.-M. Lamond A.I. et al.RRP1B targets PP1 to mammalian cell nucleoli and is associated with Pre-60S ribosomal subunits.Mol. Biol. Cell. 2010; 21: 4212-4226Crossref PubMed Scopus (35) Google Scholar, 15Bollen M. Peti W. Ragusa M.J. Beullens M. The extended PP1 toolkit: Designed to create specificity.Trends Biochem. Sci. 2010; 35: 450-458Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Of particular interest was the identification of the related Sperm antigen with calponin homology (CH) and coiled-coil domains 1 (SPECC1) and SPECC1L proteins, which were found to preferentially associate with PP1β yet possess no obvious PP1-binding motifs. This suggested that their interaction with PP1 is indirect, which was confirmed by their appearance in our MYPT1 interactome screens. MYPT1 is a regulatory subunit that binds preferentially to PP1β to generate the myosin phosphatase (MP) complex. Although most abundantly expressed in smooth muscle cells (15Bollen M. Peti W. Ragusa M.J. Beullens M. The extended PP1 toolkit: Designed to create specificity.Trends Biochem. Sci. 2010; 35: 450-458Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 16Brozovich F.V. Myosin light chain phosphatase.Circ. Res. 2002; 90: 500-502Crossref PubMed Scopus (17) Google Scholar, 17Feng J. Ito M. Ichikawa K. Isaka N. Nishikawa M. Hartshorne D.J. et al.Inhibitory phosphorylation site for rho-associated kinase on smooth muscle myosin phosphatase∗.J. Biol. Chem. 1999; 274: 37385-37390Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar), it is found in many cell types, and targeted disruption of the Mypt1 gene in mice is embryonic lethal (18Aggen J.B. Nairn A.C. Chamberlin R. Regulation of protein phosphatase-1.Chem. Biol. 2000; 7: R13-R23Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 19Okamoto R. Ito M. Suzuki N. Kongo M. Moriki N. Saito H. et al.The targeted disruption of the MYPT1 gene results in embryonic lethality.Transgenic Res. 2005; 14: 337-340Crossref PubMed Scopus (37) Google Scholar, 20Virshup D.M. Shenolikar S. From promiscuity to precision: Protein phosphatases get a makeover.Mol. Cell. 2009; 33: 537-545Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). In addition to its canonical role in regulating muscle contraction via dephosphorylation of myosin light chains, the MP complex plays a key role in the regulation of actomyosin in nonmuscle cells, affecting cell migration and adhesion (21Xia D. Stull J.T. Kamm K.E. Myosin phosphatase targeting subunit 1 affects cell migration by regulating myosin phosphorylation and actin assembly.Exp. Cell Res. 2005; 304: 506-517Crossref PubMed Scopus (66) Google Scholar). Several nonmyosin Mypt1/PP1β substrates have also been identified (for review see (22Kiss A. Erdődi F. Lontay B. Myosin phosphatase: unexpected functions of a long-known enzyme.Biochim. Biophys. Acta Mol. Cell Res. 2019; 1866: 2-15Crossref PubMed Scopus (47) Google Scholar)). They include polo-like kinase 1 (PLK1), which MYPT1 associates with at centrosomes to contribute to mitotic regulation (23Yamashiro S. Yamakita Y. Totsukawa G. Goto H. Kaibuchi K. Ito M. et al.Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1.Dev. Cell. 2008; 14: 787-797Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and Histone Deacetylase 6 (HDAC6), which plays an important role in microtubule (MT) deacetylation (24Joo E.E. Yamada K.M. MYPT1 regulates contractility and microtubule acetylation to modulate integrin adhesions and matrix assembly.Nat. Commun. 2014; 5: 1-13Crossref Scopus (47) Google Scholar). Although not studied to the same extent, depletion of SPECC1L in cells led to defects in cytoskeletal organization, cell division, and migration (25Saadi I. Alkuraya F.S. Gisselbrecht S.S. Goessling W. Cavallesco R. Turbe-Doan A. et al.Deficiency of the cytoskeletal protein SPECC1L leads to oblique facial clefting.Am. J. Hum. Genet. 2011; 89: 44-55Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 26Fan F. Roszik J. Xia L. Ghosh S. Wang R. Ye X. et al.Cytospin-A regulates colorectal cancer cell division and migration by modulating stability of microtubules and actin filaments.Cancers (Basel). 2022; 14: 1-17Crossref Scopus (3) Google Scholar), and SPECC1L mutations have been linked to developmental facial morphogenesis disorders that result in congenital malformations (25Saadi I. Alkuraya F.S. Gisselbrecht S.S. Goessling W. Cavallesco R. Turbe-Doan A. et al.Deficiency of the cytoskeletal protein SPECC1L leads to oblique facial clefting.Am. J. Hum. Genet. 2011; 89: 44-55Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Kruszka P. Li D. Harr M.H. Wilson N.R. Swarr D. McCormick E.M. et al.Mutations in SPECC1L, encoding sperm antigen with calponin homology and coiled-coil domains 1-like, are found in some cases of autosomal dominant Opitz G/BBB syndrome.J. Med. Genet. 2015; 52: 104-110Crossref PubMed Scopus (29) Google Scholar, 28Bhoj E.J. Haye D. Toutain A. Bonneau D. Nielsen I.K. Lund I.B. et al.Phenotypic spectrum associated with SPECC1L pathogenic variants: New families and critical review of the nosology of Teebi, Opitz GBBB, and baraitser-winter syndromes.Eur. J. Med. Genet. 2019; 62: 1-8Crossref PubMed Scopus (20) Google Scholar, 29Zhang T. Wu Q. Zhu L. Wu D. Yang R. Qi M. et al.A novel SPECC1L mutation causing Teebi hypertelorism syndrome: expanding phenotypic and genetic spectrum.Eur. J. Med. Genet. 2020; 63: 1-5Crossref Scopus (7) Google Scholar, 30Migliore C. Vendramin A. McKee S. Prontera P. Faravelli F. Sachdev R. et al.SPECC1L mutations are not common in sporadic cases of Opitz G/BBB syndrome.Genes (Basel). 2022; 13: 1-10Crossref Scopus (1) Google Scholar). The protein is found predominantly in the cytoplasm and has been shown to accumulate at both MT and filamentous actin structures throughout the cell cycle (25Saadi I. Alkuraya F.S. Gisselbrecht S.S. Goessling W. Cavallesco R. Turbe-Doan A. et al.Deficiency of the cytoskeletal protein SPECC1L leads to oblique facial clefting.Am. J. Hum. Genet. 2011; 89: 44-55Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 31Mattison C.P. Stumpff J. Wordeman L. Winey M. Mip1 associates with both the Mps1 kinase and actin, and is required for cell cortex stability and anaphase spindle positioning.Cell Cycle. 2011; 10: 783-793Crossref PubMed Scopus (16) Google Scholar). It is not yet clear how this distribution is regulated, as SPECC1L does not contain any obvious MT-binding domains. A single CH domain (32Gimona M. Djinovic-Carugo K. Kranewitter W.J. Winder S.J. Functional plasticity of CH domains.FEBS Lett. 2002; 513: 98-106Crossref PubMed Scopus (270) Google Scholar) at its C terminus may facilitate actin binding; however, 2 tandem CH domains are normally required to bind actin. We confirmed that SPECC1L forms a stable complex with MYPT1 in nonmuscle cells, that the binding is direct, and that it is mediated by their respective C termini. Consistent with this, quantitative proteomic experiments demonstrated significant overlap of their interaction profiles, identifying proteins involved in the regulation of cell contractility, actin organization, MT stability, junction turnover, cytokinesis, adhesion, and migration. In addition to mapping the regions of SPECC1L that mediate association with MYPT1, MTs, and actin, we also demonstrated its ability to modulate the distribution of MYPT1/PP1β between these 2 cytoskeletal networks. As part of our ongoing analysis of PP1 holoenzyme complexes, we used our quantitative SILAC (stable isotope labeling by amino acids in culture) affinity purification/mass spectrometry (AP/MS) approach (33Trinkle-Mulcahy L. Boulon S. Lam Y.W. Urcia R. Boisvert F.-M. Vandermoere F. et al.Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.J. Cell Biol. 2008; 183: 223-239Crossref PubMed Scopus (359) Google Scholar) to map the interactome of endogenous MYPT1 in U2OS cells (Fig. 1A) and compared the overlapping enriched proteins in 2 independent datasets (Fig. 1B; Supplemental Data File 1). As expected, we saw strong enrichment of both PP1β and the myosin regulatory light chain, along with numerous proteins related to motility and cytoskeletal organization. We also observed strong enrichment of SPECC1L and further confirmed their association by demonstrating coprecipitation of endogenous MYPT1 with GFP-tagged SPECC1L (Fig. 1C). An interesting observation was that not only did endogenous SPECC1L coprecipitate with transiently overexpressed GFP-tagged MYPT1 in the reciprocal experiment (Fig. S1A) but also it was the only protein besides PP1β that was significantly enriched (Fig. S1B). We had previously observed that transient overexpression of bait proteins at high levels for AP/MS-based interactome mapping can hamper incorporation into endogenous signaling complexes as binding sites saturate, although high-affinity binary interactors are usually detected (34Trinkle-Mulcahy L. Resolving protein interactions and complexes by affinity purification followed by label-based quantitative mass spectrometry.Proteomics. 2012; 12: 1623-1638Crossref PubMed Scopus (49) Google Scholar). While we were carrying out these experiments, a SPECC1L-MYPT1 association was also annotated in a large-scale screen for phosphatase interactors (13Yadav L. Tamene F. Göös H. van Drogen A. Katainen R. Aebersold R. et al.Systematic analysis of human protein phosphatase interactions and dynamics.Cell Syst. 2017; 4: 430-444.e5Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), although it was not further explored in that study. Enrichment of the related family member SPECC1 was also detected in one of the endogenous MYPT1 datasets (Supplemental Data File 1), and their association was validated by coimmunoprecipitation of MYPT1 with GFP-tagged SPECC1 (Fig. 1C). In a complementary BioID approach (35Trinkle-Mulcahy L. Recent advances in proximity-based labeling methods for interactome mapping.F1000Res. 2019; 8https://doi.org/10.12688/f1000research.16903.1Crossref PubMed Scopus (84) Google Scholar), we demonstrated biotin-based proximity labeling of MYPT1 in cells expressing either SPECC1L (Fig. 1D) or SPECC1 (Fig. 1E) fused to the biotin ligase BirA∗. We next mapped the interactome of SPECC1L in U2OS cells, to compare its overlap with that of MYPT1. As none of the available commercial antibodies were suitable for immunoprecipitation, we established a U2OS cell line stably overexpressing GFP-tagged SPECC1L at endogenous levels (Fig. 1G) and confirmed that its subcellular localization matched that of the endogenous protein (Fig. 1F, Fig. 4, A and B). Fig. 1H shows a comparison of the SPECC1L-GFP and MYPT1 interactomes. As highlighted in the Venn diagram, the high-confidence hits (top right quadrant in the graph) show strong overlap, with 76% of the SPECC1L interactors identified in the MYPT1 dataset and 43% of the MYPT1 interactors identified in the SPECC1L dataset. These overlapping factors, listed in the color-coded table (Fig. 1I), are involved in the regulation of cell contractility, actin organization, MT stability, junction turnover, cytokinesis, adhesion, and migration. A Gene Ontology Enrichment Analysis (36Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. et al.Gene Ontology: Tool for the unification of biology.Nat. Genet. 2000; 25: 25-29Crossref PubMed Scopus (28402) Google Scholar) is included in Supplemental Data File 1. Although PP1β was just below our stringent >2-fold enrichment threshold in the SPECC1L dataset, we confirmed their association by IP/WB analysis (Fig. S1C). Using GlobPlot2.3 (http://globplot.embl.de/) (37Linding R. Russell R.B. Neduva V. Gibson T.J. GlobPlot: Exploring protein sequences for globularity and disorder.Nucleic Acids Res. 2003; 31: 3701-3708Crossref PubMed Scopus (845) Google Scholar) to predict regions of disorder and globularity, we designed a truncation mutant strategy to probe for the association of specific regions of both proteins and determine whether or not their interaction is direct. We first assessed coprecipitation of endogenous MYPT1 from cell lysates with GFP-tagged SPECC1L truncation mutants (Fig. 2A). Our results indicated that the C-terminal half of SPECC1L (SPECC1L-CT; aa 462–1117) mediates its association with MYPT1, as confirmed both by IP/WB (Fig. 2B) and BioID (Fig. 2C). Similarly, the C-terminal half (aa 462–1068) of the related family member SPECC1 governs its association with MYPT1 (Fig. 2G). Further truncation of the C terminus of SPECC1L (ΔNTΔCHD) revealed that the C-terminal actin-binding CH domain is not required for MYPT1 association (Fig. 2B). To determine which region(s) of MYPT1 mediate its association with SPECC1L, we divided the protein into three fragments: an N-terminal region containing the PP1-binding RVxF motif followed by a series of ankyrin repeats (ANK; aa 1–344), a middle region (MID; aa 345–653), and a C-terminal region that contains a coiled-coil domain (CCD) and leucine zipper (LZ) domain (CD; aa 654–1030) (Fig. 2D). The localization of the fragments is consistent with previous reports that show GFP-MYPT(ANK) to be nuclear, (MID)Mypt1-GFP to be both nuclear and cytoplasmic, and GFP-Mypt1(CD) to be predominantly cytoplasmic in association with the cytoskeleton (38Wu Y. Murányi A. Erdodi F. Hartshorne D.J. Localization of myosin phosphatase target subunit and its mutants.J. Muscle Res. Cell Motil. 2005; 26: 123-134Crossref PubMed Scopus (24) Google Scholar). When affinity purified from cell lysates, only MYPT1(CD) copurified with endogenous SPECC1L (Fig. 2E). This region also mediates association with known interactors that include myosin, the active form of RhoA, the M20 MP subunit, and the inhibitory CPI-17 protein (see (22Kiss A. Erdődi F. Lontay B. Myosin phosphatase: unexpected functions of a long-known enzyme.Biochim. Biophys. Acta Mol. Cell Res. 2019; 1866: 2-15Crossref PubMed Scopus (47) Google Scholar) for review). Finally, we confirmed that, when coexpressed in cells, GFP-MYPT1(CD) copurifies with mCherry-tagged SPECC1L-CT (Fig. 2F). We next set out to determine if SPECC1L directly binds MYPT1. To do this, we first expressed and purified recombinant GST-MYPT1(CD). Recombinant SPECC1L-CT was more susceptible to degradation, so we tested all of the fragments and chose to work with SPECC1LΔCHD (1–890), which is a close representation of the full-length protein that retains MYPT1 association (Fig. 2B). Recombinant His-SPECC1LΔCHD was mixed with recombinant GST-MYPT1(CD) or GST alone, which were then captured on Glutathione Agarose beads for detection of copurified HisSpecc1LΔCHD by WB analysis using anti-His antibodies (Fig. 2H). This in vitro coprecipitation assay confirmed direct and specific interaction of His-SPECC1LLΔCHD with GST-MYPT1(CD). In a complementary far WB approach, purified His-SPECC1LΔCHD was resolved on a 1D SDS-PAGE gel, transferred to a nitrocellulose membrane, and overlaid with either purified GST-MYPT1(CD) or GST alone. Fig. 2I shows the specific and direct binding of GST-MYPT1(CD) to His-SPECC1LΔCHD. The distinct subcellular localizations observed for the various SPECC1L truncation mutants (Fig. 2A) were consistent with previous results suggesting that SPECC1L associates with both the MT network and the actin cytoskeleton (25Saadi I. Alkuraya F.S. Gisselbrecht S.S. Goessling W. Cavallesco R. Turbe-Doan A. et al.Deficiency of the cytoskeletal protein SPECC1L leads to oblique facial clefting.Am. J. Hum. Genet. 2011; 89: 44-55Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 26Fan F. Roszik J. Xia L. Ghosh S. Wang R. Ye X. et al.Cytospin-A regulates colorectal cancer cell division and migration by modulating stability of microtubules and actin filaments.Cancers (Basel). 2022; 14: 1-17Crossref Scopus (3) Google Scholar, 31Mattison C.P. Stumpff J. Wordeman L. Winey M. Mip1 associates with both the Mps1 kinase and actin, and is required for cell cortex stability and anaphase spindle positioning.Cell Cycle. 2011; 10: 783-793Crossref PubMed Scopus (16) Google Scholar). We first confirmed that the network at which the N-terminal half (SPECC1L-NT; aa 1–461) accumulates counterstained with anti-α-tubulin (Fig. 3A). This localization pattern was lost when cells were treated with the polymerization inhibitor nocodazole (NOC) to disrupt the MT network (Fig. 3A, bottom panels). We therefore concluded that this region of SPECC1L mediates MT association. In order to test whether this association is direct or indirect, we performed a MT-binding protein spin-down assay. Purified recombinant His-tagged SPECC1L-NT was mixed with freshly prepared MTs and the reaction mix centrifuged at 100,000g. At this speed, the MTs pellet along with any protein that directly associates with them. SPECC1L-NT was observed to specifically pellet in the presence of MTs and remained in the supernatant in their absence (Fig. 3C; Fig. S1D). Bovine serum albumin (BSA) was included as a negative control, as it does not bind MTs and thus remains in the soluble fraction, while the known MT-binding protein MAP4 was included as a positive control. This assay confirms, for the first time, that SPECC1L is a bona fide MT-binding protein. The presence of a CH domain (32Gimona M. Djinovic-Carugo K. Kranewitter W.J. Winder S.J. Functional plasticity of CH domains.FEBS Lett. 2002; 513: 98-106Crossref PubMed Scopus (270) Google Scholar) at the C terminus of SPECC1L (Fig. 2A) has been suggested to facilitate actin binding. Our analysis using the ELM (eukaryotic linear motif) online resource (39Kumar M. Michael S. Alvarado-Valverde J. Mészáros B. Sámano-Sánchez H. Zeke A. et al.The eukaryotic linear motif resource: 2022 release.Nucleic Acids Res. 2022; 50: D497-D508Crossref PubMed Scopus (62) Google Scholar) also identified 2 putative WH2 actin-binding domains in the C-terminal half of SPECC1L, upstream of the CH domain (Fig. 2A). We first confirmed that the structures at which SPECC1L-CT accumulates counterstain with fluorophore-tagged phalloidin, a high-affinity probe for filamentous (F)-actin (Fig. 3B). This localization pattern was lost when cells were treated with Latrunculin B (LAT), which sequesters monomeric G-actin and induces disassembly of actin filaments (Fig. 3B, bottom panels). Using a standard actin fractionation approach (40Lyubimova A. Bershadsky A.D. Ben-Ze’ev A. Autoregulation of actin synthesis responds to monomeric actin levels.J. Cell Biochem. 1997; 65: 469-478Crossref PubMed Scopus (47) Google Scholar), we further demonstrated that GFP-tagged SPECC1L-CT expressed in U2OS cells is resistant to Triton X-100 extraction, remaining in the insoluble cytoskeletal fraction with F-actin (Fig. 3D). The nonpolymerizable actin R62D mutant (41Posern G. Sotiropoulos A. Treisman R. Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor.MBoC. 2002; 13: 4167-4178Crossref Scopus (200) Google Scholar) was included to demonstrate its shift to soluble (G-actin) pools. To test the contributions of the predicted actin-binding regions to this localization, we removed either the CH domain (ΔNTΔCHD; aa 461–890) or the WH2 domains (CHD; aa 890–1117) from the C-terminal half of SPECC1L. Removal of the CH domain did not obviate actin association (Fig. 3E), although the pattern differs from that observed for the CH domain–containing fragment (Fig. 3F). While the CHD derivative distributes along straight stretches of a filamentous network, the ΔNTΔCHD mutant associates with both cortical filaments and in shorter structures in the cytoplasm. Both localization patterns are disrupted with LAT but not NOC treatment (Fig. 3, E and F, bottom panels), confirming that they represent accumulations at actin structures. This suggests that both regions play roles in the targ" @default.
• W4313856862 created "2023-01-10" @default.
• W4313856862 creator A5022905294 @default.
• W4313856862 creator A5033047541 @default.
• W4313856862 creator A5067021478 @default.
• W4313856862 creator A5070918862 @default.
• W4313856862 creator A5084733967 @default.
• W4313856862 creator A5091754610 @default.
• W4313856862 date "2023-02-01" @default.
• W4313856862 modified "2023-10-10" @default.
• W4313856862 title "SPECC1L binds the myosin phosphatase complex MYPT1/PP1β and can regulate its distribution between microtubules and filamentous actin" @default.
• W4313856862 cites W1484952182 @default.
• W4313856862 cites W1518499796 @default.
• W4313856862 cites W1966046354 @default.
• W4313856862 cites W1966554563 @default.
• W4313856862 cites W1969504295 @default.
• W4313856862 cites W1974208503 @default.
• W4313856862 cites W1977416347 @default.
• W4313856862 cites W1986041937 @default.
• W4313856862 cites W1991465744 @default.
• W4313856862 cites W1993379452 @default.
• W4313856862 cites W1995690959 @default.
• W4313856862 cites W1996221028 @default.
• W4313856862 cites W1996286498 @default.
• W4313856862 cites W2001181578 @default.
• W4313856862 cites W2015407064 @default.
• W4313856862 cites W2023214181 @default.
• W4313856862 cites W2035313584 @default.
• W4313856862 cites W2039620693 @default.
• W4313856862 cites W2055193239 @default.
• W4313856862 cites W2059093670 @default.
• W4313856862 cites W2060770087 @default.
• W4313856862 cites W2066183478 @default.
• W4313856862 cites W2072089764 @default.
• W4313856862 cites W2078636815 @default.
• W4313856862 cites W2080752012 @default.
• W4313856862 cites W2087850846 @default.
• W4313856862 cites W2093208962 @default.
• W4313856862 cites W2095910272 @default.
• W4313856862 cites W2097627784 @default.
• W4313856862 cites W2098160000 @default.
• W4313856862 cites W2103017472 @default.
• W4313856862 cites W2104726963 @default.
• W4313856862 cites W2120553816 @default.
• W4313856862 cites W2123110782 @default.
• W4313856862 cites W2123721271 @default.
• W4313856862 cites W2125812952 @default.
• W4313856862 cites W2131681216 @default.
• W4313856862 cites W2133492742 @default.
• W4313856862 cites W2145435719 @default.
• W4313856862 cites W2159447001 @default.
• W4313856862 cites W2161925427 @default.
• W4313856862 cites W2162629233 @default.
• W4313856862 cites W2166174519 @default.
• W4313856862 cites W2169898809 @default.
• W4313856862 cites W2171521148 @default.
• W4313856862 cites W2418562037 @default.
• W4313856862 cites W2577102867 @default.
• W4313856862 cites W2590493137 @default.
• W4313856862 cites W2597030906 @default.
• W4313856862 cites W2744858607 @default.
• W4313856862 cites W2763995657 @default.
• W4313856862 cites W2807269502 @default.
• W4313856862 cites W2887473550 @default.
• W4313856862 cites W2895627537 @default.
• W4313856862 cites W2901344735 @default.
• W4313856862 cites W2912096548 @default.
• W4313856862 cites W2990658682 @default.
• W4313856862 cites W2999375539 @default.
• W4313856862 cites W3016277935 @default.
• W4313856862 cites W3025924143 @default.
• W4313856862 cites W3140680224 @default.
• W4313856862 cites W3174230422 @default.
• W4313856862 cites W3183645043 @default.
• W4313856862 cites W3209420441 @default.
• W4313856862 cites W4214771551 @default.
• W4313856862 cites W4223936004 @default.
• W4313856862 doi "https://doi.org/10.1016/j.jbc.2023.102893" @default.
• W4313856862 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/36634848" @default.
• W4313856862 hasPublicationYear "2023" @default.
• W4313856862 type Work @default.
• W4313856862 citedByCount "2" @default.
• W4313856862 countsByYear W43138568622023 @default.
• W4313856862 crossrefType "journal-article" @default.
• W4313856862 hasAuthorship W4313856862A5022905294 @default.
• W4313856862 hasAuthorship W4313856862A5033047541 @default.
• W4313856862 hasAuthorship W4313856862A5067021478 @default.
• W4313856862 hasAuthorship W4313856862A5070918862 @default.
• W4313856862 hasAuthorship W4313856862A5084733967 @default.
• W4313856862 hasAuthorship W4313856862A5091754610 @default.
• W4313856862 hasBestOaLocation W43138568621 @default.
• W4313856862 hasConcept C110121322 @default.
• W4313856862 hasConcept C11960822 @default.
• W4313856862 hasConcept C125705527 @default.
• W4313856862 hasConcept C134306372 @default.
• W4313856862 hasConcept C178666793 @default.
• W4313856862 hasConcept C185592680 @default.
• W4313856862 hasConcept C20418707 @default.

• next