Abstract
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.
Highlights
The chicken tumor virus oncoprotein, v-CT10 regulator of kinase (Crk), induces a substantial increase in protein tyrosine phosphorylation through protein-protein interactions mediated by its SH22 and SH3 domains, leading to cell transformation [1]
When fibroblasts immortalized by T antigen or the 3T3 protocol were transfected with synthetic green fluorescent protein mRNA, both cell types in monolayer cultures showed evidence of green fluorescence at 1 day post transduction (DPT) (Fig. 1A)
Propidium iodide-positive cells slightly, but not significantly, increased after synthetic Cre mRNA (synCre) transfection at 3 DPT (Fig. 11, B and D). These results suggest that fibroblasts lacking Crk and CrkL undergo a modest apoptosis. Because their identification as cellular counterparts of the viral oncogene v-Crk, Crk and CrkL have been extensively studied for their roles in cell growth and tumorigenesis
Summary
Crk and CrkL Are Essential for Fibroblast Proliferation—We used the Neon system (Life Technologies) to achieve rapid and efficient gene expression in growing fibroblasts by transduction of synthetic mRNA (synRNA). To reduce innate immune responses and increase ectopic protein expression, we synthesized mRNA using the modified ribonucleotides, pseudo-UTP, and methyl-CTP [23, 24]. When fibroblasts immortalized by T antigen or the 3T3 protocol were transfected with synthetic green fluorescent protein mRNA (synGFP), both cell types in monolayer cultures showed evidence of green fluorescence at 1 day post transduction (DPT) (Fig. 1A). The intensity of fluorescence in individual cells gradually decreased.
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