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.

Upon IL-2 stimulation of T lymphocytes, the IL-2 receptor (IL-2R) becomes phosphorylated on specific tyrosine residues which serve as docking sites for proteins containing SH2 or phosphotyrosine binding domains. To study the interaction of the IL-2Rbeta chain with Shc and STAT proteins, subdomains of the IL-2Rbeta chain were expressed as tyrosine-phosphorylated glutathione S-transferase fusion proteins and used to pull-down interacting proteins from Kit 225 cell lysates. These experiments provide direct biochemical evidence that binding to the IL-2R of the adaptor protein Shc requires phosphorylation of Tyr-338 in the IL-2Rbeta acidic subdomain. In addition, we report that STAT proteins that are activated by IL-2, i.e. STAT1, STAT3 and STAT5, indeed associate with the IL-2Rbeta chain. Both the A and B isoforms of STAT5 were found to associate with Tyr-510 of the IL-2Rbeta C-terminal region, depending on its phosphorylation. In contrast, STAT1 and STAT3 associated with the IL-2Rbeta chain through its acidic subdomain. These results indicate that the interaction between IL-2Rbeta and STAT1 or 3 does not require either phosphorylation of the receptor or even the presence of tyrosine residues of IL-2Rbeta. Thus, the IL-2R recruits STAT proteins through different modes of interaction.

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Tyrosine phosphorylation of multiple proteins, including the receptor itself, is an initial event in IL-2 signaling and leads to recruitment of SH2 or PTB domain-containing proteins to the receptor. In this study, we have used subdomains of the IL-2 receptor L L chain (IL-2RL L) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56 lk , one of the critical tyrosine kinases activated by IL-2. We report that recombinant p56 lk phosphorylates in vitro tyrosine residues within the IL-2RL L chain but not those within the IL-2RQ Q chain. p56 lk phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL-2RL L chain acidic subdomain. Interestingly, phosphorylation of Tyr-358 appears to require the presence of either lk also phosphorylates very efficiently the two tyrosines present in the IL-2RL L chain C-terminal region, Tyr-392 and Tyr-510. We also investigated the association of p56 lk with the IL-2RL L chain which was found to depend on a short stretch of the IL-2RL L chain acidic subdomain, and to be independent of the presence of its tyrosine residues.z 1999 Federation of European Biochemical Societies.