We have observed that stimulation of human natural killer cells with dibutyryl cAMP (Bt2cAMP) reproduced the effects of ADP ribosylation of the GTP binding protein RhoA by Clostridium botulinum C3 transferase: both agents induced similar morphological changes, inhibited cell motility and blocked the cytolytic function. We demonstrate here that cAMP‐dependent protein kinase A (PKA) phosphorylates RhoA in its C‐terminal region, on serine residue 188. This phosphorylation does not affect the ability of recombinant RhoA to bind guanine nucleotides, nor does it modify its intrinsic GTPase activity. However, treatment of cells with Bt2cAMP results in the translocation of membrane‐associated RhoA towards the cytosol. Experiments using purified membrane preparations indicated that Rho‐GDP dissociation inhibitor, which can complex phosphorylated RhoA in its GTP‐bound state, was the effector of this translocation. Taken together, these data suggest that PKA phosphorylation of RhoA is a central event in mediating the cellular effects of cAMP, and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP‐bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP cycling.

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and a CML-like disease, as well as lymphoid leukemia, in mice. p210 BCR/ABL is an activated tyrosine kinase that phosphorylates itself and several cellular signaling proteins. The autophosphorylation site tyrosine 177 binds the adaptor Grb2 and helps determine the lineage and severity of BCR/ABL disease: Tyr177 mutation (BCR/ABL-Y177F) dramatically impairs myeloid leukemogenesis, while diminishing lymphoid leukemogenesis. The critical signal(s) from Tyr177 has remained unclear. We report that Tyr177 recruits the scaffolding adaptor Gab2 via a Grb2/Gab2 complex. Compared to BCR/ABL-expressing Ba/F3 cells, BCR/ABL-Y177F cells exhibit markedly reduced Gab2 tyrosine phosphorylation and association of phosphatidylinositol-3 kinase (PI3K) and Shp2 with Gab2 and BCR/ABL, and decreased PI3K/Akt and Ras/Erk activation, cell proliferation, and spontaneous migration. Remarkably, bone marrow myeloid progenitors from Gab2 (-/-) mice are resistant to transformation by BCR/ABL, whereas lymphoid transformation is diminished as a consequence of markedly increased apoptosis. BCR/ABL-evoked PI3K/Akt and Ras/Erk activation also are impaired in Gab2 (-/-) primary myeloid and lymphoid cells. Our results identify Gab2 and its associated proteins as key determinants of the lineage and severity of BCR/ABL transformation.

Deregulation of cell cycle checkpoints is an almost universal abnormality in human cancers and is most often due to loss-of-function mutations of tumor suppressor genes such as Rb, p53, or p16INK4a . In this study, we demonstrate that BCR/ABL inhibits the expression of a key cell cycle inhibitor, p27 Kip1 , by signaling through a pathway involving phosphatidylinositol 3-kinase (PI3K). p27Kip1 is a widely expressed inhibitor of cdk2, an essential cell cycle kinase regulating entry into S phase. We demonstrate that the decrease of p27Kip1 is directly due to BCR/ABL in hematopoietic cells by two different approaches. First, induction of BCR/ABL by a tetracycline-regulated promoter is associated with a reversible down-regulation of p27 Kip1 . Second, inhibition of BCR/ABL kinase activity with the Abl tyrosine kinase inhibitor STI571 rapidly increases p27Kip1 levels. The PI3K inhibitor LY-294002 blocks the ability of BCR/ABL to induce p27 Kip1 down-regulation and inhibits BCR/ ABL-induced entry into S phase. The serine/threonine kinase AKT/protein kinase B is a known downstream target of PI3K. Transient expression of an activated mutant of AKT was found to decrease expression of p27 Kip1, even when PI3K was inhibited by LY-294002. The mechanism of p27 Kip1 regulation is primarily related to protein stability, since inhibition of proteasome activity increased p27 Kip1 levels in BCR/ABL-transformed cells, whereas very little change in p27 transcription was found. Overall, these data are consistent with a model in which BCR/ABL suppresses p27Kip1 protein levels through PI3K/AKT, leading to accelerated entry into S phase. This activity is likely to explain in part previous studies showing that activation of PI3K was required for optimum transformation of hematopoietic cells by BCR/ABL in vitro and in vivo.