Fig. 4. CrkRS is a nuclear protein that is phosphorylated in a cell-cycle-dependent manner. (A) CrkRS is a nuclear protein. Immunoblots of cytosolic and nuclear fractions of HeLa cell lysate probed with anti-CrkRS antibodies (lanes 1,2) or precleared anti-CrkRS antibodies (lanes 3,4). (B) Anti-CrkRS immunoprecipitates have protein kinase activity. CrkRS was immunoprecipitated with anti-CRKRS antibodies and assayed for kinase activity in vitro. CrkRS (+) and an unidentified 85 kDa band (++) were phosphorylated in all anti-CrkRS immunoprecipitates (lanes 3). SR-type splicing factor ASF, casein, histone HI, myelin basic protein (MBP) and the GST-tagged CTD of yeast RNA polymerase II were added as exogenous substrates. Arrowheads indicate the position of the exogenous substrate. Kinase buffer alone (lanes 1)and anti-rabbit IgG immunoprecipitates (lanes 2) were assayed as controls. (C) CrkRS levels do not vary during the cell cycle. HeLa cells were synchronised by a thymidine/aphidicolin regime, released and collected at various times to enrich for different cell cycle phases; G₁ phase (G1), S phase (S), G₂ phase (G2), mitosis (M), and mitotic cells arrested with nocdazole (M^*). Fractions were immunoblotted with anti-CrkRS antibodies. Note that CrkRS levels do not vary but CrkRS migrates more slowly on SDS-PAGE at mitosis. (D) CrkRS is a phosphoprotein. Anti-CrkRS immunoprecipitates were treated, or not, with protein phosphatase, in the absence or presence of phosphatase inhibitors and immunoblotted with anti-CrkRS antibodies. Note that both interphase (I) and mitotic (M) forms of CrkRS shift downwards after phosphatase treatment.