Association of STAT1, STAT3 and STAT5 proteins with the IL-2 receptor involves different subdomains of the IL-2 receptor beta chain.

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.

Biochemical analysis of interleukin-2 receptor beta chain phosphorylation by p56(lck).

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 beta chain (IL-2Rbeta) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56(lck), one of the critical tyrosine kinases activated by IL-2. We report that recombinant p56(lck) phosphorylates in vitro tyrosine residues within the IL-2Rbeta chain but not those within the IL-2Rgamma chain. p56(lck) phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL-2Rbeta chain acidic subdomain. Interestingly, phosphorylation of Tyr-358 appears to require the presence of either Tyr-355 or Tyr-361. p56(lck) also phosphorylates very efficiently the two tyrosines present in the IL-2Rbeta chain C-terminal region, Tyr-392 and Tyr-510. We also investigated the association of p56(lck) with the IL-2Rbeta chain which was found to depend on a short stretch of the IL-2Rbeta chain acidic subdomain, and to be independent of the presence of its tyrosine residues.

Recent advances in the understanding of interleukin-2 signal transduction.

Interleukin-2 is one of the critical cytokines that control the proliferation and differentiation of cells of the immune system. The present article briefly reviews the current and recently established knowledge on the intracellular signaling events that convert the initial interaction of IL-2 with its receptor into pathways leading to the various biological functions. A first step in IL-2 signaling is the activation of several protein tyrosine kinases that phosphorylate a large array of intracellular substrates including the receptor complex. Phosphorylated tyrosine residues within the receptor then serve as docking sites for multimolecular signaling complexes that initiate three major pathways: the Jak-STAT pathway controlling gene transcription, the Ras-MAPK pathway leading to cell proliferation and gene transcription as well, and the PI3-kinase pathway involved in antiapoptotic signaling and organization of the cytoskeleton. Finally, other recently identified and presumably important tyrosine kinase substrates, whose significance is not yet fully understood, are described.

Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes.

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.

Signal transduction of interleukin 2 in human natural killer cells: involvement of the p56lck tyrosine kinase.

Despite numerous reports, the role of the protein tyrosine kinase p56lck in IL-2 signal transduction has remained controversial. We show here, using IL-2-dependent human natural killer cell lines, that p56lck is regulated by IL-2 in two different ways: (1) IL-2 induces a rapid increase of p56lck kinase activity as assessed in vitro; and (2) following IL-2 stimulation, p56lck undergoes phosphorylation on serine residues that is reflected by a modification of its electrophoretic mobility in SDS-PAGE. Furthermore, dose response experiments, and blocking studies performed with anti-IL-2R alpha antibodies, indicated that binding of IL-2 to the IL-2R beta chain was sufficient to produce these modifications of p56lck. In contrast, activation of the CD2 pathway stimulated the kinase activity of p56lck, but did not induce a significant shift in NK cells, as opposed to T lymphocytes. Western blot analyses, and immunoprecipitations of cell lysates from 32P-preloaded NK cells demonstrated that seven major proteins are tyrosine phosphorylated in response to IL-2. These phosphoproteins, with apparent molecular weights of 190, 150, 120, 110, 85, 65 and 56, which may not all be p56lck substrates, undergo phosphorylation and dephosphorylation with different kinetics. Furthermore, pp120 was identified as rasGAP, by Western blot and immunoprecipitation experiments. rasGAP and some of its co-precipitating molecules become phosphorylated in response to IL-2, presumably by p56lck, which would thus provide a link between IL-2R and downstream events critical for NK cell proliferation and function.