Abstract
We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.
Highlights
Durham, North Carolina 27710 and the 5-DeD. artment of Physiological Chemistry, The Ohio State University Medical Center, Columbus, Ohio 43210
The peptides were added to the indicated concentrations, and the reaction was started beled Calmodulin Binding-We investigated the influence of protein kinase C (PKC)-dependent phosphorylation of the chicken and bovine myristoylated alanine-ricCh kinase substrate (MARCKS) proteinson calmodulin binding
Cross-linkingof MARCKSProteins to Radiolabeled Calmod- ulin after phosphorylation with PKC, albeit to a lesser extent din-Preliminary studiesindicated thatboth bovine and than theunphosphorylated form (Fig. 2, compare lanes 1 and chicken MARCKS proteins could bind to calmodulin-agarose 3),in agreement with a previous report [30]. affinity columns in a calcium-dependent manner and could Cross-linking of Radiolabeled Calmodulin to the MARCKS be eluted with EGTA
Summary
North Carolina 27710 and the 5-DeD. artment of Physiological Chemistry, The Ohio State University Medical Center, Columbus, Ohio 43210. Dant cellular substrate for protein kinaCse might bea These, respectively, activateproteinkinase C (PKC)’and calmodulin-binding protein. Activation of PKC and chicken and 80,000-87,000 frombovine cellsand tissues, the myristoylated alanine-ricCh kinase substrate (MARCKS).The MARCKS proteins from bothspecies could be cross-linked to ‘251-calmodulinin a Ca2+-dependent manner. Phosphorylationof either protein by proteinkinase C prevented 1251-calmodulinbinding andcross-linking,suggestingthatthe calmodulinbinding domain mighbt e located at or near thsietes of calcium/calmodulin-dependent processes often occurs in parallel, but little is known about the interplay between these two important signal transduction pathways [3]. Because of the physical properties of these proteins,we domain inhibited calmodulin binding to theMARCKS proposed the name myristoylated alanine-rich C kinase subprotein and could be cross-linked to ‘251-calm~d- strates (MARCKS) [11].These proteins share several charulin in a calcium-dependent manner. Thesesharedproperties include 1) phoslabeled 5-dimethylaminonaphthalene-1-sulfonyl-cal-phorylation by PKC, both in cell-free systems and in intact ettdinmTihentriuhsiosehtcnuipdsilescsbleeuasa,iolbcltoiatmenyetfibhddmtouewpehdenretpinuodhpthMthltaeecieonnopaAisaf-tntlp,bmiRbdhdkihComnoeoiisiunddKdgisniamuiohnSedslcigisepcdniatcarseo-faotdCrftmilathiomeensapneiieionientccndynicao.Pwundalnahmltelisinaomtnntohdcsa,todtpnpaduhtpaneuclocrirelornthIioliyCn-blvfalbs-Saapabi2obttcsniii.lono8odoyludfnnienlinoad4ngrMfde.gc8ci,ppnyaprarncgueroMnlossoipdtecef-.o-ntstcetaMBahhellmeellaaAyclcisttantRm,rcuaooCasi2pclgeaKm)ohhcmooSiotadfrdnmepbctuoshroeolimoiesnmn,tsalepei3mflinouon)skiusnsigeimsc;athditntoitiamhlonccateinboirg(ydinihrtwisnsaiciuedgetecisrhriov,hestneowoisadenteslouebcarnidtaoenlrialgitvemchSnidpeoDiosconntdithSisehniunoig-eclpatfapik,nonptaeeldrarnybidanmagialdcntllnahuredrydetl4iyalnp)amisgnmotehiasqceitsnebduvhiaoebeeehcvuinnoliingcitdnptueeuy)eel-.s. ual activation of Ca2+-calmodulin-dependenptrocesses
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
