Highly Purified pp60srcInduces the Actin Transformation in Microinjected Cells and Phosphorylates Selected Cytoskeletal Proteins In Vitro

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Abstract
The src gene product of Rous sarcoma virus (pp60src) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60src fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60src by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60src. A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60src was achieved by this procedure. Purified pp60src phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [γ-32P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60src became labeled with 32P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60src into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60src, thus implicating pp60src as the active agent. Examination of actin-associated proteins as substrates for the pp60src kinase in vitro showed that vinculin was phosphorylated directly by pp60src, although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60src purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.