Confluence induced threonine41/serine45 phospho-beta-catenin dephosphorylation via ceramide-mediated activation of PP1cgamma.
It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101-11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-beta-catenin at threonine41/serine45. The effect of confluence on beta-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of beta-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented beta-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cgamma prevented dephosphorylation of beta-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cgamma to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced beta-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.
Marchesini
Norma
N
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 175 Ashley Ave., POB 250509, Charleston, SC 29425, USA.
Jones
Jeffrey A
JA
Hannun
Yusuf A
YA
D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013912:Threonine / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
D002518:Ceramides; D007527:Isoenzymes; D034741:RNA, Small Interfering; D051176:beta Catenin; D013912:Threonine; D012694:Serine; D054645:Protein Phosphatase 1; C491484:SMPD3 protein, human; D013108:Sphingomyelin Phosphodiesterase