Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen.

Abstract

We characterized the tyrosine phosphorylation sites of free pp6O-src and of pp60CSrc associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60`-src were different, both in vitro and in vivo. Free pp60csrc was phosphorylated in vitro at a single site, tyrosine 416. pp60Src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60CSrC in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60csrc associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60csrc associated with mT in transformed cells are identical to those of pp60(-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60Csrc associated with mT may play a role in the enhancement of the pp660csrc protein kinase activity and in cell transformation by polyomavirus.The Rous sarcoma virus transforming protein pp6ov-src and its cellular homolog pp6oc-src are membrane-associated phosphoproteins with protein-tyrosine kinase activity (9,10,14,21,23,31,33,38). The specific activity of pp6Ocsrc, when assayed in vitro with peptide substrates, is 10% or less of the specific activity of pp6Ov-src (17, 25). Phosphorylation may regulate the enzymatic activities of these two proteins: phosphorylation of amino-terminal tyrosine residues in pp6v-src is accompanied by increased protein kinase activity (3,7,11,37); phosphorylation of tyrosine in the carboxyterminal region of pp60csrc decreases its protein kinase activity (13). The major site of tyrosine phosphorylation in vivo is different in the viral and cellular protein; pp6-src is phosphorylated on tyrosine 416, whereas pp60c-src is phosphorylated on a different tyrosine residue in the carboxy-terminal region of the molecule (8,30,36,44).The middle tumor antigen (mT) of polyomavirus plays a central role in transformation (50). mT is associated with a protein-tyrosine kinase activity (20,39,45) which correlates with the transforming ability of the protein (4,20,39,45,49). mT lacks intrinsic protein kinase activity (40, 41), but associates with a known cellular protein kinase, pp6-src (2,9,15,16,33,38). pp60c-src complexed to mT shows enhanced protein kinase activity and novel sites of tyrosine phosphorylation in vitro (2,5,13,53). Because the protein kinase activity of pp6Oc-src appears to be negatively regulated by carboxy-terminal tyrosine phosphorylation (13), we examined the possibility that binding to mT might prevent phosphorylation at this site on pp6c-src, thereby increasing its protein kinase activity. We compared the phosphorylation sites of free pp60csrc with those of pp6Oc-src associated with mT.

RACK1: a novel substrate for the Src protein-tyrosine kinase

“…The purified protein-tyrosine kinases that we tested included: Src (expressed from recombinant baculovirus and purified from Sf9 cells) (Upstate Biotechnology, Lake Placid, NY, USA), c-Abl (gift from Jean Wang, University of California, San Diego, CA, USA), Epidermal Growth Factor Receptor (EGFR) (Promega, Madison, WI, USA) and Platelet-Derived Growth Factor Receptor (PDGFR) (Upstate Biotechnology). An equivalent specific activity (5 U) of purified Src, Abl, EGFR or PDGFR kinase, or Src immunoprecipitates, were incubated with 1 mg of GST -RACK1 or GST for 10 min at 308C in 30 ml of kinase buffer containing 50 mM piperazine-N-N'-bis (2-ethanesulphonic acid) (pH 7.0), 10 mM manganese chloride, 10 mM magnesium chloride, 10 mM DTT, 10 mM ATP and 25 Ci [g-32 P]ATP (4000 Ci/mmol; ICN, Costa Mesa, CA, USA) (Cartwright et al, 1985(Cartwright et al, , 1986(Cartwright et al, , 1987(Cartwright et al, , 1990Park and Cartwright, 1995). Proteins were resolved by SDS -PAGE.…”

“…For other experiments, cells were lysed in modified RIPA buffer (0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 10 mM sodium phosphate (pH 7.0), 100 mM sodium vanadate, 50 mM sodium fluoride, 50 mM leupeptin, 1% aprotinin, 2 mM EDTA and 1 mM dithiothreitol (DTT) (Cartwright et al, 1985(Cartwright et al, , 1986(Cartwright et al, , 1987(Cartwright et al, , 1990Park and Cartwright, 1995). Lysates were centrifuged at 14 000 g for 1 h at 48C.…”

Polyomavirus middle-sized tumor antigen modulates c-Jun phosphorylation and transcriptional activity.

“…MT is a membrane-associated protein that forms a complex with several cellular proteins potentially involved in growth control, including pp60c-src (2) and other members of the src family (3,4), the 85-kDa and 110-kDa subunits of phosphatidylinositol (PI) 3-kinase (5,6), and the A and C subunits of protein phosphatase 2A (7,8). MT increases the activity of pp60c-src in the complex, at least in part by preventing the phosphorylation of an inhibitory site, Tyr-527 (9,10). PI 3-kinase binds to phosphorylated tyrosine residues in a number of oncogene products and growth factor receptors, including Tyr-315 of MT (11,12).…”

The carboxy-terminal sequence of p56lck can regulate p60c-src.

“…The sequence surrounding and including Tyr-527 is absent in p6Ov-src, a transforming variant of p60csrc that has much greater protein-tyrosine kinase activity than does p60`csrc (13,15,20,32,38). Transformation by the polyomavirus middle T antigen leads to dephosphorylation of Tyr-527 and stimulation of p60csrc kinase activity (2,4,11). Alteration of c-src codon 527 to specify Phe, a residue which is not phosphorylated, activates the transforming potential of c-src (3,18,28,30).…”

“…Tyr-416 is the major site of phosphorylation in vitro (27,35). Enzymatically active forms of p60c-src are also phosphorylated at Tyr-416 in the cell (3,4,15,18,20,28,30). Tyr-527 is the major site phosphorylated in repressed forms of p60c-src in fibroblasts (7,19).…”