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pp60 c‐src mediates ERK activation/nuclear localization and PAI‐1 gene expression in response to cellular deformation
Author(s) -
Samarakoon Rohan,
Higgins Paul J.
Publication year - 2003
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.10247
Subject(s) - proto oncogene tyrosine protein kinase src , kinase , microbiology and biotechnology , mapk/erk pathway , biology , tyrosine kinase , chemistry , signal transduction
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. © 2003 Wiley‐Liss, Inc.