title: Pp60c-src mediates ERK activation/nuclear localization and PAI-1 gene expression in response to cellular deformation.
abstract: Release of transcellular tension upon disruption of actin stress fibers with cytochalasin D (CD) and associated changes in cell morphology are reflected in the rapid transcription of "deformation-responsive" genes. For certain genes (e.g., urokinase plasminogen activator and its type-1 inhibitor PAI-1), de novo mRNA synthesis appears to require cell shape-dependent activation of the MAP kinases ERK1/2. ERK activation in response to microfilament disruption was inhibited completely by the broad-spectrum tyrosine kinase inhibitor genistein and the relatively src-kinase selective compound PP1. Such inhibitor sensitivity profiles suggested that src-family members, likely pp60(c-src), were important upstream elements in deformation-related ERK activation. pp60(c-src) kinase activity was elevated fourfold within 15 min after CD addition to quiescent R22 smooth muscle cells and declined quickly thereafter. CD-induced increases in the phosphorylation levels of both pp60(c-src) and IgG heavy chain (a substrate target in the coupled immunoprecipitation/in vitro pp60(c-src) kinase assay) were ablated completely by pretreatment with the src-type kinase inhibitor PP1. Prior PP1 exposure similarly repressed CD-stimulated PAI-1 transcript accumulation. Consistent with the pharmacologic findings, transfection of a dominant-negative pp60(c-src) expression construct (DN-Src) effectively suppressed (in a concentration-dependent manner) CD-induced PAI-1 synthesis in R22 cells. To more specifically address the potential involvement of src kinases in CD-initiated ERK mobilization, R22 cells were transiently co-transfected with DN-Src and Myc-tagged ERK2 expression constructs, serum-deprived then stimulated with CD. The effect of DN-Src expression on endogenous ERK1/2 activation and nuclear translocation was assessed in separate experiments. The phosphorylation levels of both exogenous (Myc-ERK2) and endogenous ERK1/2 targets was significantly reduced by DN-Src; nuclear accumulation of pERK1/2 was completely inhibited. These data indicate that pp60(c-src) is a critical upstream activator of the ERK cascade leading to PAI-1 transcription in response to cellular deformation stimuli.
authors.affiliation: Center for Cell Biology & Cancer Research, Albany Medical College, New York 12208, USA.
authors.forename: Rohan
authors.initials: R
authors.lastname: Samarakoon
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Higgins
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis* / Q000378:metabolism*; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D004791:Enzyme Inhibitors; D017395:Plasminogen Activator Inhibitor 1; D011720:Pyrazoles; D011743:Pyrimidines; D015638:Cytochalasin D; D016392:Proto-Oncogene Proteins pp60(c-src); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases