Abstract
The Crk-associated substrate, p130(CAS), has been implicated in the regulation of the actin cytoskeleton following ligation of cell integrins with the extracellular matrix. Integrin-mediated cell adhesion involves p130(CAS) association with focal adhesion kinase (p125(FAK)). Internalization/cell entry of type 2 and type 5 adenoviruses (Ad) is also mediated by alpha(v) integrins. However, expression of dominant negative forms of p125(FAK) does not alter virus entry, and Ad entry occurs normally in p125(FAK)-deficient fibroblasts. We now provide evidence that Ad internalization, a process which is mediated by alpha(v) integrins, also requires p130(CAS) and phosphatidylinositol-3-OH kinase (PI 3-kinase). Ad induces p130(CAS) phosphorylation and inhibition of p130(CAS) phosphorylation by tyrphostin and genistein, or expression of the substrate domain deleted p130(CAS) blocks Ad internalization. p130(CAS) was also found to associate with the p85 subunit of PI 3-kinase through its proline-rich domain during virus internalization and expression of p130(CAS) containing a deleted proline-rich domain (PRD) inhibited adenovirus cell entry. We showed further that the RPLPSPP motif in the proline-rich region of p130(CAS) interacts with the SH3 domain of p85/PI 3-kinase. These studies reveal the molecular basis by which p130(CAS) coordinates the signaling pathways involved in integrin-mediated Ad endocytosis.
Highlights
The identification of cellular receptors that mediate entry of different human pathogens (Salmonella, Listeria, human immunodeficiency virus, herpes simplex virus type-1, adenovirus) has provided an opportunity to investigate the precise biochemical events involved in their pathogenesis [1, 2]
Recent genetic studies have indicated that p130CAS plays a critical role in the organization of the actin cytoskeleton. p130CAS-deficient mouse embryos die in utero and show marked cardiovascular defects which are linked to altered myofibril organization as well as defective actin stress fiber formakinase; Ad, adenovirus; p125FAK, p125 focal adhesion kinase; p130CAS, p130 Crk-associated substrate; SH2 and SH3, Src homology 2 and 3, respectively; PRD, proline-rich domain; SD, substrate domain; SB, substrate binding region; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; aa, amino acid(s)
We found that adenovirus cell entry involves p130CAS and that an association of p130CAS with PI 3-kinase but not p125FAK is crucial for this process. p130CAS interaction with p85/PI 3-kinase is mediated by an RXL motif in the proline-rich domain of p130CAS
Summary
The identification of cellular receptors that mediate entry of different human pathogens (Salmonella, Listeria, human immunodeficiency virus, herpes simplex virus type-1, adenovirus) has provided an opportunity to investigate the precise biochemical events involved in their pathogenesis [1, 2]. P130CAS-deficient mouse embryos die in utero and show marked cardiovascular defects which are linked to altered myofibril organization as well as defective actin stress fiber formakinase; Ad, adenovirus; p125FAK, p125 focal adhesion kinase; p130CAS, p130 Crk-associated substrate; SH2 and SH3, Src homology 2 and 3, respectively; PRD, proline-rich domain; SD, substrate domain; SB, substrate binding region; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; aa, amino acid(s).
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