The protein-tyrosine kinase activity of the proto-oncogene product p60c-src is negatively regulated by the phosphorylation of a tyrosine residue close to the C terminus, tyrosine 527. The phosphorylation might be catalysed by a so-far-unidentified tyrosine kinase, distinct from p60c-src. Recently we purified a protein-tyrosine kinase that specifically phosphorylates tyrosine 527 of p60c-src from neonatal rat brain. We have now confirmed the specificity of this enzyme by using a mutant p60c-src that has a phenylalanine instead of tyrosine 527, and cloned a complementary DNA that encodes the enzyme. The enzyme is similar to kinases of the src family in that it has two conserved regions, Src-homology regions 2 and 3, upstream of a tyrosine kinase domain. The amino-acid identity of each region is no more than 47%, however, and the enzyme lacks phosphorylation sites corresponding to tyrosines 416 and 527 of p60c-src and has no myristylation signal. These results suggest that this protein-tyrosine kinase, which might negatively regulate p60c-src, represents a new type of tyrosine kinase.

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Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34 cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

The product of the protooncogene c-src is a protein-tyrosine kinase, p0C-src, that is normally inhibited by phosphorylation at a tyrosine residue close to the C terminus