Regulation of pp60^c-src and its interaction with polyomavirus middle T antigen in insect cells

High yields of soluble, biologically active pp60^c-src and middle t antigen (MTAg) of polyomavirus were produced in insect cells, using a baculovirus expression system. In mammalian cells, pp60^c-src undergoes a regulatory phosphorylation on Tyr-527 in vivo and is autophosphorylated on Tyr-416 in vitro. In insect cells, pp60^c-src was phosphorylated primarily on Tyr-416, although Tyr-527 was detectable at a low level. A kinase-negative mutant of pp60^c-src was not phosphorylated on either Tyr-527 or Tyr-416 in bisect cells and thus is an excellent biochemical reagent to search for the regulatory kinase that usually phosphorylates Tyr-527 in mammalian cells. MTAg synthesized in insect cells was not phosphorylated on tyrosine residues in vivo or in vitro, suggesting that it did not associate with any endogenous tyrosine kinases. However, MTAg isolated from cells coinfected with viruses encoding both MTAg and pp60^c-src was phosphorylated on tyrosine residues both in vivo and in vitro.