Cdk4 integrates growth stimulatory and inhibitory signals during G1 phase of hematopoietic cells.

Proliferation of hematopoietic cells is controlled by both growth stimulatory and inhibitory cytokines acting primarily in G1, but the mechanisms which integrate these disparate signals are unknown. In a myeloid cell line dependent on interleukin-3 (IL-3) for proliferation, expression of the cyclin dependent kinase Cdk4 and D-type cyclin partners, D2 and D3, in mid G1 was found to be directly related to the concentration of IL-3. TGF beta 1, which induces cell cycle arrest in mid-G1, blocked IL-3-induced expression of Cdk4, but had no effect on expression of cyclins D2 or D3. Sublines made to constitutively express Cdk4, but not lines constitutively expressing cyclins D2 or D3, were hyper responsive to IL-3 and resistant to TGF beta 1. Using an in vitro kinase assay with recombinant retinoblastoma protein (Rb) as a substrate, cyclin D2-associated kinase activity was shown to be induced in G1 by IL-3 and inhibited by TGF beta 1. Constitutive expression of Cdk4, but not cyclin D2 or D3, increased cyclin D2-associated Rb kinase activity and this activity could no longer be inhibited by TGF beta 1. Also, in vivo phosphorylation of Rb was inhibited by TGF beta 1 in wild type but not in Cdk4 lines. Cdk2 kinase activity was also decreased by TGF beta 1, and restored by overexpression of Cdk4. These results implicate Cdk4 activity as a mid G1 checkpoint sensitive to both growth stimulatory and inhibitory cytokines.