Fibroblast Growth Requires CT10 Regulator of Kinase (Crk) and Crk-like (CrkL).
CT10 regulator of kinase (Crk) and Crk-like (CrkL) are the cellular counterparts of the viral oncogene v-Crk Elevated levels of Crk and CrkL have been observed in many human cancers; inhibition of Crk and CrkL expression reduced the tumor-forming potential of cancer cell lines. Despite a close relationship between the Crk family proteins and tumorigenesis, how Crk and CrkL contribute to cell growth is unclear. We ablated endogenous Crk and CrkL from cultured fibroblasts carrying floxed alleles of Crk and CrkL by transfection with synthetic Cre mRNA (synCre). Loss of Crk and CrkL induced by synCre transfection blocked cell proliferation and caused shrinkage of the cytoplasm and the nucleus, formation of adherens junctions, and reduced cell motility. Ablation of Crk or CrkL alone conferred a much more modest reduction in cell proliferation. Reintroduction of CrkI, CrkII, or CrkL individually rescued cell proliferation in the absence of the endogenous Crk and CrkL, suggesting that Crk and CrkL play overlapping functions in regulating fibroblast growth. Serum and basic FGF induced phosphorylation of Akt, MAP kinases, and S6 kinase and Fos expression in the absence of Crk and CrkL, suggesting that cells lacking Crk and CrkL are capable of initiating major signal transduction pathways in response to extracellular stimuli. Furthermore, cell cycle and cell death analyses demonstrated that fibroblasts lacking Crk and CrkL become arrested at the G1-S transition and undergo a modest apoptosis. Taken together, our results suggest that Crk and CrkL play essential overlapping roles in fibroblast growth.
From the Children's Research Institute, Children's Mercy Kansas City, Kansas City, Missouri 64108 tjpark@cmh.edu.
Taeju
http://orcid.org/0000-0003-3897-3994
T
Park
From the Children's Research Institute, Children's Mercy Kansas City, Kansas City, Missouri 64108.
Mateusz
http://orcid.org/0000-0002-3857-6633
M
Koptyra
From the Children's Research Institute, Children's Mercy Kansas City, Kansas City, Missouri 64108.
Tom
http://orcid.org/0000-0003-1444-7551
T
Curran
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk / Q000235:genetics / Q000378:metabolism*; D016196:S Phase / Q000187:drug effects / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; C476798:Crk protein, mouse; D009687:Nuclear Proteins; D050737:Proto-Oncogene Proteins c-crk; D016222:Fibroblast Growth Factor 2; D051057:Proto-Oncogene Proteins c-akt; D020929:Mitogen-Activated Protein Kinase Kinases
adaptor protein; apoptosis; cell cycle; cell growth; fibroblast; growth factor; serum