The Role of CKIP-1 in Cell Morphology Depends on Its Interaction with Actin-capping Protein

CKIP-1 is a pleckstrin homology domain-containing protein that induces alterations of the actin cytoskeleton and cell morphology when expressed in human osteosarcoma cells. CKIP-1 interacts with the heterodimeric actin-capping protein in cells, so we postulated that this interaction was responsible for the observed cytoskeletal and morphological effects of CKIP-1. To test this postulate, we used peptide “walking arrays” and alignments of CKIP-1 with CARMIL, another CP-binding protein, to identify Arg-155 and Arg-157 of CKIP-1 as residues potentially required for its interactions with CP. CKIP-1 mutants harboring Arg-155 and Arg-157 substitutions exhibited greatly decreased CP binding, while retaining wild-type localization, the ability to interact with protein kinase CK2, and self-association. To examine the phenotype associated with expression of these mutants, we generated tetracycline-inducible human osteosarcoma cells lines expressing R155E,R157E mutants of CKIP-1. Examination of these cell lines reveals that CKIP-1 R155E,R157E did not induce the distinct changes in cell morphology and the actin cytoskeleton that are characteristic of wild-type CKIP-1 demonstrating that the interaction between CKIP-1 and CP is required for these cellular effects. CKIP-1 is a pleckstrin homology domain-containing protein that induces alterations of the actin cytoskeleton and cell morphology when expressed in human osteosarcoma cells. CKIP-1 interacts with the heterodimeric actin-capping protein in cells, so we postulated that this interaction was responsible for the observed cytoskeletal and morphological effects of CKIP-1. To test this postulate, we used peptide “walking arrays” and alignments of CKIP-1 with CARMIL, another CP-binding protein, to identify Arg-155 and Arg-157 of CKIP-1 as residues potentially required for its interactions with CP. CKIP-1 mutants harboring Arg-155 and Arg-157 substitutions exhibited greatly decreased CP binding, while retaining wild-type localization, the ability to interact with protein kinase CK2, and self-association. To examine the phenotype associated with expression of these mutants, we generated tetracycline-inducible human osteosarcoma cells lines expressing R155E,R157E mutants of CKIP-1. Examination of these cell lines reveals that CKIP-1 R155E,R157E did not induce the distinct changes in cell morphology and the actin cytoskeleton that are characteristic of wild-type CKIP-1 demonstrating that the interaction between CKIP-1 and CP is required for these cellular effects. CKIP-1 4The abbreviations used are: CKIP-1, CK2-interacting protein 1; CK2, protein kinase CK2 or casein kinase II; CP, actin-capping protein; GFP, green fluorescent protein; mAb, monoclonal antibody; GST, glutathione S-transferase; DMF, N,N-dimethylformamide; ITC, isothermal titration calorimetry; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate. was identified in a yeast two-hybrid screen for novel interaction partners of protein kinase CK2 (1Bosc D.G. Graham K.C. Saulnier R.B. Zhang C. Prober D. Gietz R.D. Litchfield D.W. J. Biol. Chem. 2000; 275: 14295-14306Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The cDNA for CKIP-1 codes for a protein of ∼46 kDa with an amino-terminal pleckstrin homology (PH) domain and a carboxyl-terminal leucine-rich region as well as five putative PXXP motifs. The PH domain of CKIP-1 is required for phospholipid binding in vitro and for plasma membrane localization in cells (1Bosc D.G. Graham K.C. Saulnier R.B. Zhang C. Prober D. Gietz R.D. Litchfield D.W. J. Biol. Chem. 2000; 275: 14295-14306Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 2Olsten M.E. Canton D.A. Zhang C. Walton P.A. Litchfield D.W. J. Biol. Chem. 2004; 279: 42114-42127Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Furthermore, this domain is necessary for interactions with protein kinase CK2, because mutants lacking the PH domain fail to interact with the kinase. Additionally, we have demonstrated that a subpopulation of protein kinase CK2 is targeted to the plasma membrane by CKIP-1 in cells (2Olsten M.E. Canton D.A. Zhang C. Walton P.A. Litchfield D.W. J. Biol. Chem. 2004; 279: 42114-42127Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). This targeting of CK2 is lost when the PH domain of CKIP-1 is replaced by a myristoylation recognition sequence, even though the CKIP-1 mutant still localizes to the plasma membrane. These results suggest that CKIP-1 may function in an analogous manner to protein kinase A anchoring proteins, which target cAMP-dependent protein kinase A (3Colledge M. Dean R.A. Scott G.K. Langeberg L.K. Huganir R.L. Scott J.D. Neuron. 2000; 27: 107-119Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 4Dodge K. Scott J.D. FEBS Lett. 2000; 476: 58-61Crossref PubMed Scopus (117) Google Scholar, 5Alto N. Carlisle Michel J.J. Dodge K.L. Langeberg L.K. Scott J.D. Diabetes. 2002; 51: S385-S388Crossref PubMed Google Scholar, 6Wong W. Scott J.D. Nat. Rev. Mol. Cell. Biol. 2004; 5: 959-970Crossref PubMed Scopus (857) Google Scholar). In addition to this potential role as a CK2-targeting protein, CKIP-1 appears to have roles independent of CK2. Recent reports have shown that CKIP-1 functions in muscle cell differentiation (7Safi A. Vandromme M. Caussanel S. Valdacci L. Baas D. Vidal M. Brun G. Schaeffer L. Goillot E. Mol. Cell. Biol. 2004; 24: 1245-1255Crossref PubMed Scopus (52) Google Scholar) and AP-1 regulation and apoptosis (8Zhang L. Xing G. Tie Y. Tang Y. Tian C. Li L. Sun L. Wei H. Zhu Y. He F. EMBO J. 2005; 24: 766-778Crossref PubMed Scopus (57) Google Scholar). To investigate the cellular functions of CKIP-1, we generated cell lines with tetracycline-regulated expression of FLAG-CKIP-1. Induction of FLAG-CKIP-1 in these cells caused changes in cellular morphology, as well as increases in F-actin and total cellular levels of actin (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). To determine the mechanistic basis for these observations, we performed a proteomic screen using Tandem affinity purification (10Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1423) Google Scholar) and large-scale immunoprecipitations to identify CKIP-1 interaction partners. We identified the heterodimeric actincapping protein as a novel CKIP-1-interacting protein (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). Moreover, we showed that CKIP-1 can partially inhibit the activity of CP at the barbed ends of actin filaments. Collectively, these observations suggested two hypothetical models. First, it is that the interaction of CP with CKIP-1 at the plasma membrane binding of CP to the barbed ends of the actin to actin and changes in cellular the effects of CKIP-1 cell morphology may interactions with CP to target CKIP-1 to the barbed ends of actin filaments. To between these we used peptide “walking arrays” and alignments with the CP-binding protein, C. M. M. M. Cooper J.A. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google to identify Arg-155 and Arg-157 as residues of CKIP-1 potentially required for its binding with CP. To investigate the of these residues for interactions between CKIP-1 and CP, we mutants of CKIP-1 harboring substitutions at Arg-155 and Arg-157 as well as a peptide the putative CP-binding region of CKIP-1. to test changes in cell morphology and the actin cytoskeleton by CKIP-1 its interactions with CP, we generated human osteosarcoma cell lines expressing CP-binding mutants of CKIP-1 the of the and actin green fluorescent protein and and of actin-capping protein D.A. Cooper J.A. J. Biol. PubMed Scopus Google Scholar) the the of the of and by the of of CKIP-1 and in a (1Bosc D.G. Graham K.C. Saulnier R.B. Zhang C. Prober D. Gietz R.D. Litchfield D.W. J. Biol. Chem. 2000; 275: 14295-14306Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). a of of was generated by and and as (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). CKIP-1 to interactions with CP generated for using the as by the FLAG-CKIP-1 was generated using the and the FLAG-CKIP-1 was generated using the and the CKIP-1 generated using the and the CKIP-1 R155E,R157E generated using the and the CKIP-1 generated using the and the using CKIP-1 as a by CKIP-1 cells or cells in and The with and at in a for at The and with and protein performed with or as a with to protein for at protein in the of and cells in of and or The and as The to using to protein or to protein for at In the and the protein with the the the by the addition of by using the of PubMed Scopus Google Scholar). to for at A using a performed to the using at and to performed to the using at and to performed by for in in with by with at with to with was performed by for in in by with using to and performed by for in in by with at of and In used at a of with for J. Methods. 2002; PubMed Scopus Google Scholar, Mol. Biol. Google Scholar) was in the of peptide for in the residues of CKIP-1 required for interaction with CP. with a of at a of in a of to of with the addition of in to of the of peptide of of the of the membrane with a of in with with in with with and of in a of and for the of the in the peptide the membrane was in a to the for was the membrane in the in for with with with and with a of of CP was using a expression for of and or and a Y. H. S. K. J. PubMed Scopus Google Scholar). CP was expressed and to as S. Cooper J.A. J. Biol. 2001; PubMed Scopus Google Scholar). CP was used for and peptide CP was used for in vitro actin to the the membrane was with and The peptide was for in in by with CP at with the was with CP at a of CP was with to a isothermal titration Methods. PubMed Scopus Google Scholar). The heterodimeric was as S. Cooper J.A. J. Biol. 2001; PubMed Scopus Google Scholar). CP and CKIP-1 peptide and CP and CKIP-1 peptide to and using the was and was to CP protein was the cell and CKIP-1 peptide was the of the CKIP-1 peptide was performed at with an by with a of The cell was at the The performed the showed of binding The and of binding by of the CP CKIP-1 to a with the was using The results of two independent are F-actin as in Li F. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). of actin in was in the of F-actin CP, and of CKIP-1 protein and CKIP-1 and human was for at The of the CKIP-1 and and In and FLAG-CKIP-1 and FLAG-CKIP-1 R155E,R157E by in vitro and using a with to the or expressed in and using as D.G. E. C. Litchfield D.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in vitro and performed by of the with of and of or was at for with The by the was and the with of by the addition of and to a the using a and for two independent using of cells the human osteosarcoma cell and the tetracycline-regulated protein C. S. C. D.A. EMBO J. PubMed Scopus Google of a cell with tetracycline-regulated expression of FLAG-CKIP-1 (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). lines expressing R155E,R157E generated by cells with mutant and in the of with and and to of FLAG-CKIP-1 cells and for expression by in with at in an of with and for in at for with in and with for with in with and with at for at with PBS, to for in the with and with a and of cells, with and for in at with with for in the with PBS, and with using a and F-actin performed by the of A. J. Biol. PubMed Scopus Google Scholar). of cells with and of F-actin with and and for at The cells by and with of and The was in of by and levels by the at at to the protein and the of two independent of CP with have shown that expression of CKIP-1 induces alterations in cell morphology and the actin The of the heterodimeric in with Tandem affinity and FLAG-CKIP-1 suggested that interactions have a role in the observed morphological and cytoskeletal alterations (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). Examination of of CKIP-1 and CP in cells CKIP-1 shown to differentiation and in human osteosarcoma cells CKIP-1 In this is in of and cells, is not with or with protein A These observations the of of CP-binding have demonstrated using that interaction between CKIP-1 and CP is of CKIP-1 (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar) in To identify residues required for this interaction we generated a CKIP-1 peptide of the to of this with of between peptide The was with CP by with shown in two exhibited interactions with CP. A of the CP-binding domain of CKIP-1 reveals that and to well between a by C. M. M. M. Cooper J.A. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrated that the interaction of with CP two and was that and motifs. we performed an of with the region of CKIP-1 and in the with the that the residues necessary for interactions between and CP in CKIP-1. Moreover, the residues in CKIP-1 and are between of between CKIP-1 and the in we performed to determine of or of the CP-binding CKIP-1 in of To which with the CP-binding region of CARMIL, we generated in which residues Arg-155 and Arg-157 to or we because this was between CKIP-1 and The CKIP-1 and we generated CKIP-1 and These are shown in To between these two binding we these interactions with CP by cells with FLAG-CKIP-1 as and the with to performed these using by with of R155E,R157E in putative in greatly binding to CP as did or not CKIP-1 harboring substitutions or substitutions not in binding to CP. These results that of Arg-155 and Arg-157 CKIP-1 is to interaction with CP, substitutions not To CKIP-1 with substitutions we the effects of binding to CP using and cells with FLAG-CKIP-1 and the these cells with to The with and with with wild-type CKIP-1, mutants greatly binding to CP. performed with the CKIP-1 mutants greatly binding to CP. To the results of the we and glutathione and with to protein expression The used to with CP In with the results mutants greatly binding to CP. Collectively, these suggest that and of CKIP-1 are required for interaction with CP and that CKIP-1 and may CP-binding for CP with CKIP-1 interactions between CKIP-1 and CP, we a peptide to the of CKIP-1 responsible for interactions with CP as in two independent the for this interaction was to the to a binding this with for the interactions between CP and N. S. D. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) as well as interactions between CP and another CP-binding protein with a to that in CKIP-1 and S. Kim K. H. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (63) Google Scholar). is that in the by S. Kim K. H. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (63) Google the CKIP-1 peptide performed at as well as analogous and in interactions between and CP, an with the that CKIP-1 a CP-binding with these Collectively, these the of interactions between CKIP-1 and CP and the of these of the CP-binding of the ability of CP to the barbed ends of actin (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). and of CKIP-1 in the CP-binding region showed decreased We the the ability of CKIP-1 to inhibit the activity of CP in actin for actin was with F-actin the actin to of barbed of wild-type CKIP-1 the activity of CP A and at with results (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). In the R155E,R157E and CKIP-1 mutants the activity of CP the that these CKIP-1 mutants not interact with CP. We the of the CP-binding region of CKIP-1 in with the CKIP-1 peptide First, we that addition of the peptide a the activity of CP. We to a of peptide we at CP activity at to We not the for the to peptide we that this may a of the the of the In the of CKIP-1, addition of the peptide the of CKIP-1 the activity of CP. CKIP-1 and the peptide at in this so the results are with the peptide with CKIP-1 for binding CP. the peptide and CKIP-1 with of CP, have an the These results are with the CP-binding region of CKIP-1 for binding CP and a role in the interaction of CKIP-1 with CP. of CKIP-1 in test of CP-binding the cellular localization of CKIP-1, we the localization of the CKIP-1 mutants in cells cells with FLAG-CKIP-1 as and with for by with wild-type CKIP-1 localizes to the plasma membrane in cells (1Bosc D.G. Graham K.C. Saulnier R.B. Zhang C. Prober D. Gietz R.D. Litchfield D.W. J. Biol. Chem. 2000; 275: 14295-14306Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 2Olsten M.E. Canton D.A. Zhang C. Walton P.A. Litchfield D.W. J. Biol. Chem. 2004; 279: 42114-42127Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). mutants that CP binding to have cellular localization to wild-type CKIP-1. results when cells with These results that of residues to interactions with CP the cellular localization of CKIP-1. the mutants we CKIP-1 to used to CKIP-1 R155E,R157E the to with CK2 and to the of this CKIP-1 we to examine interactions between CKIP-1 and CK2 as well as of CKIP-1. the PH domain of CKIP-1 is responsible for interactions with protein kinase CK2 (2Olsten M.E. Canton D.A. Zhang C. Walton P.A. Litchfield D.W. J. Biol. Chem. 2004; 279: 42114-42127Abstract Full Text Full Text PDF PubMed Scopus (56) Google of Arg-155 and which of this not to interactions with CK2. to test that the R155E,R157E have not interactions between CKIP-1 and protein kinase CK2, wild-type FLAG-CKIP-1 and FLAG-CKIP-1 R155E,R157E to in vitro and in the of in with or and by and using a wild-type and mutant CKIP-1 the ability to interact with protein kinase CK2. To these the for two independent using and expressed as of protein These suggest that the two CKIP-1 protein kinase CK2 to the by and have suggested that CKIP-1 can to or in cells (2Olsten M.E. Canton D.A. Zhang C. Walton P.A. Litchfield D.W. J. Biol. Chem. 2004; 279: 42114-42127Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, L. Xing G. Tie Y. Tang Y. Tian C. Li L. Sun L. Wei H. Zhu Y. He F. EMBO J. 2005; 24: 766-778Crossref PubMed Scopus (57) Google Scholar). To examine the R155E,R157E have we performed immunoprecipitations to of CKIP-1 cells with with or FLAG-CKIP-1 and the these cells with to expression The with and with wild-type CKIP-1 and the R155E,R157E mutant are to interact with CKIP-1. Collectively, these that of interaction between CKIP-1 and CP not the ability of CKIP-1 to interact with protein kinase CK2 or with that of interactions between CP and CKIP-1 can of CKIP-1. of FLAG-CKIP-1 results in and that the CKIP-1 R155E,R157E mutant is an to between for the effects of CKIP-1 cell morphology, because it to interact with CP of wild-type CKIP-1. we generated cell lines with tetracycline-regulated expression of FLAG-CKIP-1 these FLAG-CKIP-1 R155E,R157E was the of the and and for expression of mutant FLAG-CKIP-1 in the of cells expressing wild-type CKIP-1 or CKIP-1 R155E,R157E in the or of and with and by cell lines and regulation of CKIP-1 To examine protein cells in the of or for These to with is and cell lines to levels of FLAG-CKIP-1. To protein the membrane was and with To determine the phenotype associated with expression of CKIP-1 we used to examine cells in the or of for we have expression of wild-type CKIP-1 in the of cells to and morphology with cells (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). of CKIP-1 R155E,R157E expression to in To these observations, we performed of the cell lines in the or of by the of cells of The results this that cells to wild-type CKIP-1 have a cells, of CKIP-1 R155E,R157E expression cell FLAG-CKIP-1 R155E,R157E or we a in and increases in cellular actin levels associated with CKIP-1 expression (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). these we of CKIP-1 R155E,R157E the by the F-actin of these cells using The wild-type and mutant CKIP-1 cell lines in the or of and with Induction of FLAG-CKIP-1 caused an in with of actin and actin with (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). In of FLAG-CKIP-1 R155E,R157E to in the actin To these we performed of binding In with the for wild-type CKIP-1 to protein, a in the was not observed of FLAG-CKIP-1 These that of wild-type FLAG-CKIP-1 results in an in the of cellular of a CKIP-1 mutant that to interact with CP we the total cellular levels of actin in these cell in the of or for expression of FLAG-CKIP-1 results in a in the cellular levels of actin (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). of FLAG-CKIP-1 R155E,R157E expression to a The membrane was and with to protein the of actin levels by and to levels two independent that actin levels are by in cells expressing was in FLAG-CKIP-1 R155E,R157E cells. these suggest that interaction with CP is necessary for CKIP-1 to levels of total and F-actin in cells. In cells, actin by the addition of actin to barbed ends of the To barbed ends are by in a Cooper J.A. PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. H. M. S. H. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and manner K. W. J. Biol. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, H. M. PubMed Scopus Google Scholar, Cooper J.A. PubMed Scopus Google Scholar). to the Cell. Full Text Full Text PDF PubMed Scopus Google barbed ends of actin are which actin to barbed ends the membrane. the the of and increases the of the This is by the observations that of protein caused an in F-actin a in C. Cooper J.A. Cell. Full Text PDF PubMed Scopus Google and with and demonstrating that when protein is of the was D. PubMed Scopus Google Scholar). to the regulation of actin an of is by that can interact with and the activity of CP. To to interact with CP N. S. D. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. M. N. E. S. S. N. M. Y. 2005; PubMed Scopus Google C. M. M. M. Cooper J.A. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, G. K. J.A. J. Biol. 2001; PubMed Scopus Google Scholar, K. M. A. J.A. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google S. Cooper J.A. EMBO J. 2004; PubMed Scopus Google N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google H. A. N. A. J. 2005; PubMed Scopus Google and CKIP-1 (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). and CARMIL, that CKIP-1 the activity of CP in a manner C. M. M. M. Cooper J.A. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The that the of by CKIP-1 is not at that the of CKIP-1 with CP a decreased affinity for barbed ends that binding is not In this a with of CP activity at C. M. M. M. Cooper J.A. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the of CP with CKIP-1 or in to barbed ends of actin in cells or not the of the in a which may by effects that expression of CKIP-1 cell morphology and the it is that expression of in human cells was shown to the and of C. M. M. M. Cooper J.A. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, of caused a in F-actin and in that by expression of wild-type not lacking a region in binding CP. These suggest that CARMIL, interactions with CP, can cell morphology and the actin results the that CKIP-1 and or a CP-binding as suggested by S. Kim K. H. Cooper J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (63) Google Scholar). This that CKIP-1 is a of a of that can the activity of protein, and actin and cell We two for the role of CKIP-1 in cell suggested that the interaction of CP with CKIP-1 at the plasma membrane binding of CP to the barbed ends of the actin to the of a at the plasma membrane and to actin and changes in cellular the of CP by CKIP-1 is (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar) the in which the effects of CKIP-1 cell morphology targeting of CKIP-1 to the barbed ends of actin by CP. results that of binding to CP did not the localization of CKIP-1 in cells, the In the of CP-binding did CKIP-1 to the ability to the actin cytoskeleton and the of cells, the This hypothetical is the of of CP activity to an in barbed ends an in This is with reports in the expression of CP mutants that fail to interact with actin in to an in F-actin K. A. Y. Cooper J.A. J. Biol. 2004; PubMed Scopus Google Scholar). In a as of CP D. or L. in the actin cytoskeleton as did of the protein in human cells C. M. M. M. Cooper J.A. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). these suggest that of CP to of actin in in demonstrated that CK2 at and that CK2 increases the activity of CKIP-1 CP, this activity appears to (9Canton D.A. Olsten M.E. Kim K. Doherty-Kirby A. Lajoie G. Cooper J.A. Litchfield D.W. Mol. Cell. Biol. 2005; 25: 3519-3534Crossref PubMed Scopus (63) Google Scholar). In the it of to the role of CK2 in the regulation of CP and the role of CKIP-1 in the of this In addition to the role of CKIP-1 and its interactions with CP in the regulation of cell morphology and the this a for We have shown that the morphology and the in F-actin are interactions between CKIP-1 and CP, because these changes are by the interactions CKIP-1 and CP. In this these have to an of CKIP-1 the actin cytoskeleton interactions with CP. was performed at the the of and at the of was performed in the of was performed in the and and protein purification in the at the of We are to for the of this to for with ITC, for and to and for the CKIP-1 peptide and CP,

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