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CLP-36 PDZ-LIM Protein Associates with Nonmuscle α-Actinin-1 and α-Actinin-4

2000; Elsevier BV; Volume: 275; Issue: 15 Linguagem: Inglês

10.1074/jbc.275.15.11100

ISSN

1083-351X

Autores

Tea Vallenius, Keijo Luukko, Tomi P. Mäkelä,

Tópico(s)

Cardiac Valve Diseases and Treatments

Resumo

The PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36) has been suggested to act as adapters that direct LIM-binding proteins to the cytoskeleton. Most interactions of PDZ-LIM proteins with the cytoskeleton have been identified in striated muscle, where several PDZ-LIM proteins are predominantly expressed. By contrast, CLP-36 mRNA is expressed in several nonmuscle tissues, and here we demonstrate high expression of CLP-36 in epithelial cells by in situ hybridization analysis. Our subcellular localization studies indicate that in nonmuscle cells, CLP-36 protein localizes to actin stress fibers. This localization is mediated via the PDZ domain of CLP-36 that associates with the spectrin-like repeats of α-actinin. Interestingly, immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis indicate that both nonmuscle α-actinin-1 and α-actinin-4 form complexes with CLP-36. The high expression of α-actinin-4 in the colon, together with these results, suggests a specific function for the α-actinin-4-CLP-36 complex in the colonic epithelium. More generally, results presented here demonstrate that the association of PDZ-LIM proteins with the cytoskeleton extends to the actin stress fibers of nonmuscle cells. The PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36) has been suggested to act as adapters that direct LIM-binding proteins to the cytoskeleton. Most interactions of PDZ-LIM proteins with the cytoskeleton have been identified in striated muscle, where several PDZ-LIM proteins are predominantly expressed. By contrast, CLP-36 mRNA is expressed in several nonmuscle tissues, and here we demonstrate high expression of CLP-36 in epithelial cells by in situ hybridization analysis. Our subcellular localization studies indicate that in nonmuscle cells, CLP-36 protein localizes to actin stress fibers. This localization is mediated via the PDZ domain of CLP-36 that associates with the spectrin-like repeats of α-actinin. Interestingly, immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis indicate that both nonmuscle α-actinin-1 and α-actinin-4 form complexes with CLP-36. The high expression of α-actinin-4 in the colon, together with these results, suggests a specific function for the α-actinin-4-CLP-36 complex in the colonic epithelium. More generally, results presented here demonstrate that the association of PDZ-LIM proteins with the cytoskeleton extends to the actin stress fibers of nonmuscle cells. glutathioneS-transferase polyacrylamide gel electrophoresis PDZ and LIM domains are protein interaction motifs that are found in various proteins associated with the cytoskeleton (reviewed in Refs.1.Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar and 2.Fanning A.S. Anderson J.M. J. Clin. Invest. 1999; 103: 767-772Crossref PubMed Scopus (401) Google Scholar). Recent characterization of several related proteins has revealed a new family of proteins that contain an N-terminal PDZ domain and one or more C-terminal LIM domains (3.Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar, 4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). ALP (5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar), RIL (7.Kiess M. Scharm B. Aguzzi A. Hajnal A. Klemenz R. Schwarte-Waldhoff I. Schafer R. Oncogene. 1995; 10: 61-68PubMed Google Scholar), and CLP-36 (8.Wang H. Harrison-Shostak D.C. Lemasters J.J. Herman B. Gene. 1995; 165: 267-271Crossref PubMed Scopus (46) Google Scholar) each contain a single LIM domain, whereas Enigma/LMP-1 (10.Boden S.D. Liu Y. Hair G.A. Helms J.A. Hu D. Racine M. Nanes M.S. Titus L. Endocrinology. 1998; 139: 5125-5134Crossref PubMed Scopus (0) Google Scholar,11.Wu R.Y. Gill G.N. J. Biol. Chem. 1994; 269: 25085-25090Abstract Full Text PDF PubMed Google Scholar), ENH (12.Kuroda S. Tokunaga C. Kiyohara Y. Higuchi O. Konishi H. Mizuno K. Gill G.N. Kikkawa U. J. Biol. Chem. 1996; 271: 31029-31032Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), and ZASP/Cypher1 (4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) each have three C-terminal LIM domains. In addition, two more distantly related proteins, namely LIMK-1 (13.Mizuno K. Okano I. Ohashi K. Nunoue K. Kuma K. Miyata T. Nakamura T. Oncogene. 1994; 9: 1605-1612PubMed Google Scholar) and LIMK-2 (14.Okano I. Hiraoka J. Otera H. Nunoue K. Ohashi K. Iwashita S. Hirai M. Mizuno K. J. Biol. Chem. 1995; 270: 31321-31330Crossref PubMed Scopus (170) Google Scholar), contain a kinase domain C-terminal to a single PDZ and two LIM domains. PDZ-LIM proteins have been suggested to act as adapters between kinases and the cytoskeleton (3.Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). This is based on two lines of studies indicating that PDZ-LIM proteins associate on one hand to the cytoskeleton via their PDZ domain (3.Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar, 4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), and on the other hand to kinases via their LIM domains (6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 11.Wu R.Y. Gill G.N. J. Biol. Chem. 1994; 269: 25085-25090Abstract Full Text PDF PubMed Google Scholar, 12.Kuroda S. Tokunaga C. Kiyohara Y. Higuchi O. Konishi H. Mizuno K. Gill G.N. Kikkawa U. J. Biol. Chem. 1996; 271: 31029-31032Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 15.Durick K. Wu R.Y. Gill G.N. Taylor S.S. J. Biol. Chem. 1996; 271: 12691-12694Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The LIM-kinase interaction has been demonstrated with three PDZ-LIM proteins, mostly using yeast two-hybrid screens. Enigma was identified by virtue of association of its third LIM domain to the cytoplasmic tail of the insulin receptor; this interaction is apparently important for endocytosis of the receptor (11.Wu R.Y. Gill G.N. J. Biol. Chem. 1994; 269: 25085-25090Abstract Full Text PDF PubMed Google Scholar). The association of the second LIM domain of Enigma with Ret/ptc2 is required for the plasma membrane localization and mitogenic activity of Ret/ptc2 (15.Durick K. Wu R.Y. Gill G.N. Taylor S.S. J. Biol. Chem. 1996; 271: 12691-12694Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 16.Durick K. Gill G.N. Taylor S.S. Mol. Cell. Biol. 1998; 18: 2298-2308Crossref PubMed Scopus (80) Google Scholar). ENH was discovered through its binding to protein kinase C (12.Kuroda S. Tokunaga C. Kiyohara Y. Higuchi O. Konishi H. Mizuno K. Gill G.N. Kikkawa U. J. Biol. Chem. 1996; 271: 31029-31032Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), and subsequently, Cypher1 was also found to associate with protein kinase C (6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The association of the LIM domain of RIL with the second and fourth PDZ domains of protein tyrosine phosphatase PTP-BL in a yeast two-hybrid screen (17.Cuppen E. Gerrits H. Pepers B. Wieringa B. Hendriks W. Mol. Biol. Cell. 1998; 9: 671-683Crossref PubMed Scopus (127) Google Scholar) suggests that the LIM domain interactions of PDZ-LIM proteins may not be limited to kinases. The association of PDZ domains with the cytoskeleton in PDZ-LIM proteins has been mostly studied in muscle due to their high specific expression in this tissue (3.Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar, 4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Enigma, ALP, and ZASP/Cypher1 proteins localize to the Z line of striated muscle. This localization is mediated by the association of Enigma PDZ domain with β-tropomyosin (3.Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar) or by the association of ALP and ZASP/Cypher1 PDZ domains with α-actinin-2 (4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). α-Actinin-2 and α-actinin-3 are the muscle-specific α-actinins forming part of the contractile machinery anchoring actin thin filaments at the Z lines and dense bodies in striated and smooth muscle, respectively (18.Endo T. Masaki T. J. Cell Biol. 1984; 99: 2322-2332Crossref PubMed Scopus (53) Google Scholar). Cellular α-actinin exists as an antiparallel dimer with a globular head domain, spectrin-like repeats and EF-hands (reviewed in Ref. 19.Blanchard A. Ohanian V. Critchley D. J. Muscle Res. Cell Motil. 1989; 10: 280-289Crossref PubMed Scopus (318) Google Scholar). Dimerization of α-actinin is mediated by the spectrin-like repeats of α-actinin (20.Imamura M. Endo T. Kuroda M. Tanaka T. Masaki T. J. Biol. Chem. 1988; 263: 7800-7805Abstract Full Text PDF PubMed Google Scholar, 21.Flood G. Kahana E. Gilmore A.P. Rowe A.J. Gratzer W.B. Critchley D.R. J. Mol. Biol. 1995; 252: 227-234Crossref PubMed Scopus (41) Google Scholar, 22.Djinovic-Carugo K. Young P. Gautel M. Saraste M. Cell. 1999; 98: 537-546Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). This rod domain mediates the association with the PDZ domain of ALP, whereas ZASP binds to a 155-amino acid C-terminal region of α-actinin-2 (5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar,6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). In addition to the muscle-specific α-actinins, two nonmuscle human α-actinin isoforms, α-actinin-1 (23.Youssoufian H. McAfee M. Kwiatkowski D.J. Am. J. Hum. Genet. 1990; 47: 62-71PubMed Google Scholar) and α-actinin-4 (24.Honda K. Yamada T. Endo R. Ino Y. Gotoh M. Tsuda H. Yamada Y. Chiba H. Hirohashi S. J. Cell Biol. 1998; 140: 1383-1393Crossref PubMed Scopus (391) Google Scholar), have been identified and represent independent genes. α-Actinin-1 is localized along stress fibers and takes part in bundling actin filaments (25.Lazarides E. Burridge K. Cell. 1975; 6: 289-298Abstract Full Text PDF PubMed Scopus (411) Google Scholar). It also associates with several cytoskeletal and membrane associated proteins, such as integrins (26.Otey C.A. Pavalko F.M. Burridge K. J. Cell Biol. 1990; 111: 721-729Crossref PubMed Scopus (654) Google Scholar), intercellular adhesion molecules (27.Carpen O. Pallai P. Staunton D.E. Springer T.A. J. Cell Biol. 1992; 118: 1223-1234Crossref PubMed Scopus (264) Google Scholar), N-methyl-d-aspartate receptor (28.Wyszynski M. Lin J. Rao A. Nigh E. Beggs A.H. Craig A.M. Sheng M. Nature. 1997; 385: 439-442Crossref PubMed Scopus (519) Google Scholar), and vinculin (29.Wachsstock D.H. Wilkins J.A. Lin S. Biochem. Biophys. Res. Commun. 1987; 146: 554-560Crossref PubMed Scopus (133) Google Scholar). The subcellular localization of nonmuscle α-actinin-4 differs from that of α-actinin-1 in that α-actinin-4 is less clearly concentrated to stress fibers and is not detected in focal adhesions or cell contacts (24.Honda K. Yamada T. Endo R. Ino Y. Gotoh M. Tsuda H. Yamada Y. Chiba H. Hirohashi S. J. Cell Biol. 1998; 140: 1383-1393Crossref PubMed Scopus (391) Google Scholar). Moreover, localization is cell type-dependent, sometimes demonstrating nuclear staining (24.Honda K. Yamada T. Endo R. Ino Y. Gotoh M. Tsuda H. Yamada Y. Chiba H. Hirohashi S. J. Cell Biol. 1998; 140: 1383-1393Crossref PubMed Scopus (391) Google Scholar). Unlike α-actinin-1, the expression of α-actinin-4 is induced in migrating cells (24.Honda K. Yamada T. Endo R. Ino Y. Gotoh M. Tsuda H. Yamada Y. Chiba H. Hirohashi S. J. Cell Biol. 1998; 140: 1383-1393Crossref PubMed Scopus (391) Google Scholar). During studies on proteins associated with a partially characterized novel kinase, 1T. Vallenius and T. P. Mäkelä, unpublished data. we became interested in the CLP-36 PDZ-LIM protein. CLP-36 (also called hCLIM1; Ref. 9.Kotaka M. Ngai S.M. Garcia-Barcelo M. Tsui S.K. Fung K.P. Lee C.Y. Waye M.M. J. Cell. Biochem. 1999; 72: 279-285Crossref PubMed Scopus (43) Google Scholar) was initially identified as a rat gene down-regulated during hypoxia in hepatocytes (8.Wang H. Harrison-Shostak D.C. Lemasters J.J. Herman B. Gene. 1995; 165: 267-271Crossref PubMed Scopus (46) Google Scholar).CLP-36 mRNA is expressed in several nonmuscle tissues (8.Wang H. Harrison-Shostak D.C. Lemasters J.J. Herman B. Gene. 1995; 165: 267-271Crossref PubMed Scopus (46) Google Scholar, 9.Kotaka M. Ngai S.M. Garcia-Barcelo M. Tsui S.K. Fung K.P. Lee C.Y. Waye M.M. J. Cell. Biochem. 1999; 72: 279-285Crossref PubMed Scopus (43) Google Scholar), and here we demonstrate high expression of CLP-36 in epithelial cells by in situ hybridization analysis. Moreover, CLP-36 was found to localize to actin stress fibers via its PDZ domain through its association with cellular α-actinin-1 and α-actinin-4, suggesting that CLP-36 acts as an adapter between stress fibers and LIM-binding proteins in nonmuscle cells. Embryos of CBA × NMRI mice at stages E7, E9, E11, E15, and E17.5 were timed by both vaginal plugs of mothers and by morphological criteria. The experiments were approved by the Animal Welfare Committee of the Haartman Institute, University of Helsinki. Tissue preparation and in situ hybridization using a antisense or sense RNA probe generated from human CLP-36cDNA (nucleotides 87–1504 in GenBankTM accession number U90878) were performed as described (30.Luukko K. Moshnyakov M. Sainio K. Saarma M. Sariola H. Thesleff I. Dev. Dyn. 1996; 206: 87-99Crossref PubMed Scopus (79) Google Scholar). The Myc-tagged CLP-36 plasmid was generated by subcloning anEcoRI-XhoI fragment from 38/JG4–5 (nucleotides 87–1504 in GenBankTM accession number U90878) intoEcoRI-SalI sites of pAMC (31.Tiainen M. Ylikorkala A. Makela T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9248-9251Crossref PubMed Scopus (264) Google Scholar). Myc-tagged CLPΔ1–24 was done similarly using 51/JG4–5 (nucleotides 212–1504 in GenBankTM accession number U90878). U2OS osteosarcoma or COS-7 cells were transfected by using the calcium phosphate transfection method as described (32.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Mouse monoclonal (9E10) or rabbit polyclonal (PRB-150C) antibodies against Myc epitopes were from Babco Inc. (Berkeley, CA). The following α-actinin antibodies were used: A5044 mouse monoclonal antibody to detect α-actinin-1 (Sigma), NCC-Lu-632 mouse monoclonal antibody to detect α-actinin-4 (a kind gift of Dr. Hirohashi; Ref. 24.Honda K. Yamada T. Endo R. Ino Y. Gotoh M. Tsuda H. Yamada Y. Chiba H. Hirohashi S. J. Cell Biol. 1998; 140: 1383-1393Crossref PubMed Scopus (391) Google Scholar), and a rabbit polyclonal α-actinin antibody (33.Fujiwara K. Porter M.E. Pollard T.D. J. Cell Biol. 1978; 79: 268-275Crossref PubMed Scopus (122) Google Scholar). Transfected cells on coverslips were fixed with 3.5% (w/v) paraformaldehyde, permeabilized with 0.1% Triton X-100 for 5 min, labeled with antibodies or Hoechst, and analyzed as described (31.Tiainen M. Ylikorkala A. Makela T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9248-9251Crossref PubMed Scopus (264) Google Scholar). The 0.5% Triton X-100 extraction or the 1-h cytochalasin B treatment prior to fixation were performed as described (34.Sainio M. Zhao F. Heiska L. Turunen O. den Bakker M. Zwarthoff E. Lutchman M. Rouleau G.A. Jaaskelainen J. Vaheri A. Carpen O. J. Cell Sci. 1997; 110: 2249-2260Crossref PubMed Google Scholar). Immunoprecipitations were performed as described (31.Tiainen M. Ylikorkala A. Makela T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9248-9251Crossref PubMed Scopus (264) Google Scholar) except that the cleared supernatants in ELB (150 mm NaCl, 50 mmHEPES, pH 7.4, 5 mm EDTA, 0.1% Nonidet P-40, 1 mm dithiothreitol, 2.5 μg/ml aprotinin, 0.5 mm phenylmethylsulfonyl fluoride, 10 mmβ-glycerol-phosphate, 1 μg/ml leupeptin) were incubated with 1 μl of monoclonal anti-Myc antibody for 2 h at +4 °C prior to 1 h immunoprecipitation. Western blotting analysis was according to standard procedures (32.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For expression of GST-CLP-36,2 aNotI fragment from 38/pAMC was subcloned into pAcGHLT-A (Pharmingen, San Diego, CA) baculovirus transfer vector, which was introduced into Sf9 insect cells together with BaculoGold baculovirus DNA as recommended by the manufacturer. For protein expression, the baculovirus was propagated in Hi5 insect cells (Invitrogen) for 48 h. The GST-CLP-36 protein was purified using glutathione-Sepharose and eluted with glutathione. The bacterially produced chicken GST-α-actinin proteins (a kind gift from Dr. Critchley; Ref. 35.Salmikangas P. Mykkanen O.M. Gronholm M. Heiska L. Kere J. Carpen O. Hum. Mol. Genet. 1999; 8: 1329-1336Crossref PubMed Scopus (171) Google Scholar) were purified as described (32.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For detection of association between cellular proteins and GST-CLP-36, 200 μg of COS-7 cell extracts in ELB were incubated with the indicated amounts of GST-CLP-36 for 2 h at +4 °C prior to adding 7.5 μl (packed volume) of glutathione-Sepharose beads for 1 h. Subsequently, the beads were washed four times with ELB and subjected to SDS-PAGE and Coomassie staining (see Fig. 6). For mapping of the CLP-36-α-actinin association, 200 μg of transfected COS-7 cell lysate in ELB were incubated with 4 μg of GST-α-actinins for 2 h at +4 °C prior to anti-Myc immunoprecipitation, SDS-PAGE, and Western blotting analysis (see Fig. 4 B).Figure 4α-Actinin associates with CLP-36 via its spectrin-like repeats. A, Coomassie stain of bacterial GST-actinin fragments indicated on the right and used as input for the experiment in B. GST-ABD/R1-R2 contains the actin-binding domain and spectrin-like repeats 1 and 2; GST-R1-R4 contains all four spectrin-like repeats; GST-R3-R4/EF contains spectrin-like repeats 3 and 4 and the EF-hand. B,to map the binding site of α-actinin to CLP-36, COS-7 cell extracts expressing Myc-CLP-36 were incubated with GST-α-actinin fragments. Subsequently, anti-Myc immunoprecipitates were analyzed by Western blotting analysis with anti-α-actinin (α-actinin) to detect associated GST-α-actinin proteins and cellular α-actinin or with anti-Myc (Myc) to control the levels of CLP-36.View Large Image Figure ViewerDownload (PPT) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed on a BiflexTM time-of-flight instrument equipped with a nitrogen laser operating at 337 nm. CLP-36 associated α-actinin band from either U2OS or COS7 cells was cut out from the Coomassie-stained gel, digested with trypsin, eluted, and analyzed in the linear positive ion delayed extraction mode. The output was analyzed using ProFound. Northern blot analyses of CLP-36 indicated expression in various tissues with some variability between rat (8.Wang H. Harrison-Shostak D.C. Lemasters J.J. Herman B. Gene. 1995; 165: 267-271Crossref PubMed Scopus (46) Google Scholar) and human (9.Kotaka M. Ngai S.M. Garcia-Barcelo M. Tsui S.K. Fung K.P. Lee C.Y. Waye M.M. J. Cell. Biochem. 1999; 72: 279-285Crossref PubMed Scopus (43) Google Scholar) tissues. To determine the pattern of expression ofCLP-36 in adult tissues and during embryogenesis, in situ hybridization analysis with an antisense CLP-36probe was performed. Hybridization of a sagittal section of a E11 mouse embryo (Fig. 1 A) indicated expression in several tissues, including dorsal root ganglia, liver, and arteries. Prominent expression was also detected throughout the skin epithelium (Fig. 1 A, SE). Analysis of earlier stage embryos indicated that the skin epithelium had a strong hybridization signal even in E9 stage embryos, in which the skin is at the single layered ectodermal stage (not shown). At stage E17.5 expression in the skin and the oral cavity epithelium (Fig. 1, B andC, OC) localized predominantly to the basal cell layer, whereas expression in the outer dental epithelium was low (Fig.1, B and C, DE). CLP-36 expression was also noted in the mucosal epithelium of the developing gut from embryonal stage E15 onwards (data not shown) and in adult intestine, as demonstrated in strong epithelial expression in the small intestine (Fig. 1, D and E, IE), whereas CLP-36 expression was undetectable in the surrounding smooth muscle layers (SM). In adult mouse tissues, a prominent expression was also noted in the epithelium of esophagus and urinary bladder, whereas expression in skeletal muscle was undetectable. The expression of CLP-36 in epithelium and other nonmuscle tissues prompted us to study its subcellular localization in cell lines of nonmuscle origin. To this end, a plasmid expressing a Myc epitope-tagged CLP-36 (Myc-CLP-36) or vector control was transiently transfected into U2OS osteosarcoma (Fig.2) or COS-7 green monkey kidney epithelial cells (not shown). Double fluorescence analysis with anti-Myc and rhodamine-labeled phalloidin from vector transfected or Myc-CLP-36 transfected cells indicated partial colocalization of CLP-36 with filaments resembling actin stress fibers (Fig. 2 A). To verify this colocalization, similar coverslips were subjected to detergent extraction or treated with cytochalasin B prior to fixation. As demonstrated in Fig. 2 B (Triton X-100), detergent extraction revealed colocalization of CLP-36 and actin stress fibers. If microfilament formation was inhibited by cytochalasin B prior to fixation, CLP-36 was partially accumulated with the disrupted actin-containing filaments (Fig. 2 B, cytochalasin B). These experiments indicate that a significant fraction of CLP-36 is associated with actin stress fibers. During the immunoprecipitation studies, it was noted that whenever Myc-CLP-36 was purified from cell lysates, a prominent associated protein of approximately 100 kDa was detected when staining for total protein. The size of the polypeptide together with the immunofluorescence pattern of CLP-36 suggested that the band represented α-actinin. Moreover, based on interactions of the other PDZ-LIM proteins (3.Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar, 4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), the association was further predicted to be mediated by the PDZ domain of CLP-36. To test this hypothesis, immunoprecipitates from cells expressing either Myc-CLP-36 or a mutant lacking part of the PDZ domain (Myc-CLPΔ1–24) were analyzed by Western blotting with an α-actinin antibody (33.Fujiwara K. Porter M.E. Pollard T.D. J. Cell Biol. 1978; 79: 268-275Crossref PubMed Scopus (122) Google Scholar). α-Actinin was only detected in the lane immunoprecipitated with the full-length CLP-36 (Fig. 3 A), indicating that the first 24 amino acids of CLP-36 are required for α-actinin binding and thus suggesting that an intact PDZ domain is required for binding. The inability of Myc-CLPΔ1–24 to associate with α-actinin was associated with loss of localization to stress fibers (Fig.3 B). This indicates that CLP-36 localization to stress fibers is mediated through its association with α-actinin. Previously PDZ-LIM proteins have been reported to associate with two distinct regions of α-actinin (4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar). Therefore, the interaction site of α-actinin with CLP-36 was mapped using three fragments of α-actinin-1 fused to GST. GST-ABD/R1-R2 contains the actin-binding domain and spectrin-like repeats 1 and 2; GST-R1-R4 contains all four spectrin-like repeats; GST-R3-R4/EF contains spectrin-like repeats 3 and 4 and the EF-hand. Bacterially produced proteins (Fig. 4 A) were incubated with Myc-CLP-36 expressing or control cell extracts. Following anti-Myc immunoprecipitation and SDS-PAGE, the presence of Myc-CLP-36 (Fig. 4 B, Myc) and of copurified α-actinin fragments was analyzed (Fig. 4 B, α-actinin). The results indicated the presence of significant amounts of GST-R1-R4 in CLP-36 immunoprecipitates. Low levels GST-ABD/R1-R2 were also detected specifically in CLP-36 immunoprecipitates. The presence of GST-R3-R4/EF was due to unspecific binding, as it was also detected in the control lane. Association of the endogenous α-actinin with CLP-36 was also noted in all cases, as expected (Fig. 4 B, α-actinin). However, the level of associated α-actinin decreased in the presence of GST-R1-R4 indicating competitive binding of these with CLP-36. As endogenous α-actinin and GST-R1-R4 were detected using the same antibody, the results also demonstrated that the total amount of CLP-36-associated GST-R1-R4 was significantly higher than CLP-36-associated endogenous α-actinin. This indicates that association of GST-R1-R4 with CLP-36 is not mediated indirectly through dimerization with endogenous α-actinin. As the levels of CLP-36-associated GST-ABD/R1-R2 were lower than those of endogenous α-actinin, the same conclusion cannot be made for GST-ABD/R1-R2. Taken together, these results indicate that the efficient association of CLP-36 with α-actinin is mediated via the spectrin-like repeats. The expression of CLP-36 in nonmuscle cells suggested that it could associate with either α-actinin-1 or the closely related α-actinin-4. To investigate this, double fluorescence analysis of U2OS cells expressing Myc-CLP-36 was performed with anti-Myc and either A5044 to detect α-actinin-1 or NCC-Lu-632 to detect α-actinin-4. As shown in Fig. 5 A, Myc-CLP-36 was colocalized both with α-actinin-1 and α-actinin-4 specifically in stress fibers and was more clearly detected after Triton X-100 pretreatment. CLP-36 was not detected in areas of focal adhesions. In addition, anti-Myc immunoprecipitates from Myc-CLP-36 expressing cells contained both α-actinin-1 and α-actinin-4 (Fig.5 B). To estimate the stoichiometry of binding of α-actinin to CLP-36, varying amounts of the GST-CLP-36 protein were incubated with 200 μg of COS-7 cell lysate and subsequently purified using glutathione-Sepharose beads and analyzed with a Coomassie stain of SDS-PAGE (Fig. 6). A prominent band of approximately 100 kDa representing α-actinin was the only protein detected in addition to GST-CLP-36. An approximate mass ratio of 2:3 for GST-CLP-36 and α-actinin was linear until α-actinin in the lysate became limiting. Considering the molecular masses of GST-CLP-36 (66 kDa) and α-actinin (103 kDa) this mass ratio indicates an equimolar complex, and together with the higher affinity of CLP-36 to actinin dimers, it suggests that two molecules of CLP-36 are associated with an α-actinin dimer. The efficient purification of soluble α-actinin from the cell extracts using GST-CLP-36 also enabled analysis of this protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis, which indicated the presence of both α-actinin-1 and α-actinin-4 in the 100-kDa band. The present study demonstrates that nonmuscle α-actinin-1 and α-actinin-4 associate with the PDZ domain of CLP-36 in actin stress fibers. This, together with the abundant expression ofCLP-36 in skin and intestinal epithelia and other nonmuscle tissues, implicates a role for it as an adapter between stress fibers and LIM-binding proteins in these tissues. The high expression of α-actinin-4 in the colon and in epithelial cells (24.Honda K. Yamada T. Endo R. Ino Y. Gotoh M. Tsuda H. Yamada Y. Chiba H. Hirohashi S. J. Cell Biol. 1998; 140: 1383-1393Crossref PubMed Scopus (391) Google Scholar) suggests a specific function for the α-actinin-4-CLP-36 complex in the colonic epithelium. The expression pattern observed in the mouse in situhybridization analyses is in concordance with previous Northern blotting data from rat tissues (8.Wang H. Harrison-Shostak D.C. Lemasters J.J. Herman B. Gene. 1995; 165: 267-271Crossref PubMed Scopus (46) Google Scholar), both indicating thatCLP-36 is not expressed in skeletal muscle. However, a positive signal in Northern blotting analysis from human skeletal muscle (9.Kotaka M. Ngai S.M. Garcia-Barcelo M. Tsui S.K. Fung K.P. Lee C.Y. Waye M.M. J. Cell. Biochem. 1999; 72: 279-285Crossref PubMed Scopus (43) Google Scholar) suggests either species-specific expression differences or the presence of a closely related human gene. Analysis of expressed sequence tag data base content did not reveal closely related cDNAs, and 8 of 244 (3.3%) human CLP-36 expressed sequence tags were from skeletal muscle, supporting low relative expression of human CLP-36 in this tissue. Previous studies have implicated roles for PDZ-LIM proteins in skeletal muscle. This is based both on the muscle-oriented approaches used in prior studies (3.Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar, 4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) and also on the predominant expression ofALP and ZASP/Cypher in skeletal muscle by Northern blotting analysis (4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). However, results from this study, as well as from those with Enigma (11.Wu R.Y. Gill G.N. J. Biol. Chem. 1994; 269: 25085-25090Abstract Full Text PDF PubMed Google Scholar), ENH (12.Kuroda S. Tokunaga C. Kiyohara Y. Higuchi O. Konishi H. Mizuno K. Gill G.N. Kikkawa U. J. Biol. Chem. 1996; 271: 31029-31032Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 36.Ueki N. Seki N. Yano K. Masuho Y. Saito T. Muramatsu M. J. Hum. Genet. 1999; 44: 256-260Crossref PubMed Scopus (31) Google Scholar), and RIL (7.Kiess M. Scharm B. Aguzzi A. Hajnal A. Klemenz R. Schwarte-Waldhoff I. Schafer R. Oncogene. 1995; 10: 61-68PubMed Google Scholar, 37.Bashirova A.A. Markelov M.L. Shlykova T.V. Levshenkova E.V. Alibaeva R.A. Frolova E.I. Gene. 1998; 210: 239-245Crossref PubMed Scopus (29) Google Scholar), indicate that the PDZ-LIM family is similarly involved in mediating interactions of LIM-binding proteins with the cytoskeleton in other tissue types. In this regard, it is also interesting to note that the mRNA expression of RIL is concentrated in the epithelial cells (7.Kiess M. Scharm B. Aguzzi A. Hajnal A. Klemenz R. Schwarte-Waldhoff I. Schafer R. Oncogene. 1995; 10: 61-68PubMed Google Scholar). However, there is a notable difference in tissue distribution between RIL and CLP-36. RIL is highly expressed in the epithelium of brain, testis, mid-size bronchii, uterine tubae and stomach, whereas CLP-36 mRNA expression in these tissues is low or undetectable. Although the subcellular localization and the PDZ domain associated protein of RIL have not been identified, it is interesting to speculate that these two close relatives might share a common function specific for a particular type of epithelium. An extension of this hypothesis includes ALP, the third close relative of CLP-36 and RIL, with a nonoverlapping expression pattern (5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar). ALP could mediate a similar or related function in skeletal muscle. CLP-36 was predominantly localized to stress fibers in an apparently PDZ domain-dependent fashion. CLP-36 also directly associated with α-actinin in an apparently PDZ domain-dependent fashion, indicating that CLP-36 localization to stress fibers is mediated via α-actinin. Interaction of CLP-36 with α-actinin is not limited to stress fibers, as CLP-36 efficiently coimmunoprecipitated with cellular α-actinin from nonionic, low detergent cell lysates. Analysis of solubleversus insoluble fraction of such lysates indicated that roughly equal amounts of both α-actinin and CLP-36 are present in soluble and insoluble fractions (data not shown). Based on these results, it is interesting to speculate that the soluble α-actinin may represent a free pool to be used for rapid reorganization of actin stress fibers in nonmuscle cells and that the associated CLP-36 could be involved in this function. CLP-36 associated efficiently with an α-actinin fragment containing all four spectrin-like repeats. Previously, all four repeats were demonstrated to be required for a maximally stable α-actinin dimer (21.Flood G. Kahana E. Gilmore A.P. Rowe A.J. Gratzer W.B. Critchley D.R. J. Mol. Biol. 1995; 252: 227-234Crossref PubMed Scopus (41) Google Scholar, 22.Djinovic-Carugo K. Young P. Gautel M. Saraste M. Cell. 1999; 98: 537-546Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Taken together, these results suggest that CLP-36 associates with α-actinin dimers. This fits well with the observed stoichiometry of the CLP-36-α-actinin complex, indicating that two molecules of CLP-36 are associated with an α-actinin dimer. The identification of PDZ domain interactions has revealed the association of several PDZ-LIM proteins with the microfilaments (3.Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar, 4.Faulkner G. Pallavicini A. Formentin E. Comelli A. Ievolella C. Trevisan S. Bortoletto G. Scannapieco P. Salamon M. Mouly V. Valle G. Lanfranchi G. J. Cell Biol. 1999; 146: 465-476Crossref PubMed Scopus (188) Google Scholar, 5.Xia H. Winokur S.T. Kuo W.L. Altherr M.R. Bredt D.S. J. Cell Biol. 1997; 139: 507-515Crossref PubMed Scopus (187) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). This has led to the suggestion that the primary function of these proteins is to act as adapters between the cytoskeleton and LIM-binding proteins (3.Guy P.M. Kenny D.A. Gill G.N. Mol. Biol. Cell. 1999; 10: 1973-1984Crossref PubMed Scopus (91) Google Scholar, 6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). So what are these LIM-binding proteins? The identified interactions of some LIM domains with serine/threonine kinases (6.Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 12.Kuroda S. Tokunaga C. Kiyohara Y. Higuchi O. Konishi H. Mizuno K. Gill G.N. Kikkawa U. J. Biol. Chem. 1996; 271: 31029-31032Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) suggests that similar partners may be found for the orphan LIM domains as well. This is supported by our original identification of the LIM domain of CLP-36 in a yeast two-hybrid screen using a novel partially characterized serine/threonine kinase.2 This would point to a role for CLP-36 in recruiting kinase(s) to actin stress fibers via α-actinin binding. We thank Nisse Kalkkinen for mass spectrometry analysis; Birgitta Tjäder for technical assistance; and Drs. O. Carpen, S. Hirohashi, and D. Critchley for reagents. The Mäkelä laboratory (especially Päivi Ojala) is acknowledged for fruitful discussions.

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