Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells.

Abstract

T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p2l1 in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p2l'as, and it has been proposed that the TCR effect on p21r' occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p2l', a permeabilized cell system was used to examine TCR regulation of p2l1 under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p2l1 activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p2l'-GTP complexes, and this stimulation correlated with an inactivation of p2l' GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKCmediated, TCR-induced stimulation of p2l'. These data indicate that two mechanisms for p21l'S regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p2l1 via a non-PKC-controlled route that may involve tyrosine kinases.The three ras proto-oncogenes, Ha-, Ki-, and N-ras, encode 21,000-molecular-weight guanine nucleotide-binding proteins that cause cell transformation when constitutively activated by point mutation (3). The activity of p2lras is normally regulated by a cycle of binding GTP to give the biologically active form of the protein followed by hydrolysis of bound GTP to GDP (22). The GDP-bound form of p2lras is inactive and is reactivated by exchange of bound GDP for free cytosolic GTP. Recent studies have shown that in T lymphocytes, triggering of the T-cell antigen receptor (TCR), the adhesion molecule CD2, or the interleukin 2 (IL-2) receptor caused a very rapid stimulation of p2lras, as measured by its conversion from the GDP-to the GTP-bound state in activated cells (9,11,12). Ras proteins can also be regulated in fibroblasts by signals generated by the receptors for platelet-derived growth factor, epidermal growth factor, and insulin (5,29,30). In mast cells, p2lras is activated by the cytokines IL-3 and granulocyte macrophage colony-stimulating factor (31).In T lymphocytes, phorbol esters that activate protein kinase C (PKC) can mimic TCR triggering and induce accumulation of p21ras-GTP complexes (9). The TCR is known to stimulate PKC by a mechanism involving the generation of diacylglycerols (DAGs; the endogenous PKC activators) via phospholipase C (PLC)-mediated hydrolysis of inositol phospholipids (PtdIns) (17,27). TCR-stimulated PKC could thus mediate the p2lras activation seen upon triggering of this receptor. The proposal that PKC functions as an upstream regulator of p2lras in T cells is in contrast to the previously described link between p2lras and PKC in fibroblasts, where, based on observations that "active" * Corresponding author.

Positive regulation of human T cell activation by Gi2 proteins and interleukin-8

“…In other systems, signaling through G i2 proteins and tyrosine kinases converge on the MAP kinase (ERK) activation pathway, possibly at the level of Shc or Grb2 via tyrosine kinases of the Src family [7,8]. Thus, the ERK pathway, which, along with Src kinases Lck and Fyn, must be activated for TCR-induced IL-2 production [9,41,42], could be the pathway regulated by G i2 proteins in T lymphocytes. In summary, we have shown that in human T cells, G i2 proteins are required to modulate metabolic pathways leading to CD25/CD69 up-regulation, IL-2 production, and cell proliferation in response to TCR activation.…”

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

“…This leaves open the possibility that a GEF other than Sos is responsible for at least some of the Ras activation stimulated by antigen receptors. Part of the Ras activation stimulated by the T-cell receptor may also be due to Ras GAP inhibition rather than GEF activation (17,35). Lymphocytes have a Ras activation pathway that is initiated by phorbol ester treatment, and this is only partially or not at all dependent on tyrosine kinase activity (17,35).…”

“…Part of the Ras activation stimulated by the T-cell receptor may also be due to Ras GAP inhibition rather than GEF activation (17,35). Lymphocytes have a Ras activation pathway that is initiated by phorbol ester treatment, and this is only partially or not at all dependent on tyrosine kinase activity (17,35). Most of this phorbol ester-stimulated activation of Ras seems to be mediated by PKCs (35), but it is certainly possible that Ras-GRP could also contribute, perhaps with a dependence on concurrent PKC activity.…”

The Lck SH3 Domain Is Required for Activation of the Mitogen-activated Protein Kinase Pathway but Not the Initiation of T-cell Antigen Receptor Signaling

“…Tyrosine phosphorylation of the TCR complex occurs within amino acid sequences known as immunoreceptor-based tyrosine activation motifs (ITAMs) and is required for receptor signaling function (4 -6). Activation of downstream signaling pathways, including the Ras/mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol (PIP 2 ) pathway, and the phosphatidylinositol 3-kinase (PI-3-K) pathway, is dependent upon proteintyrosine kinase function (7)(8)(9)(10). However, the mechanisms by which the induction of tyrosine kinase activity initiates these diverse signaling processes is unknown.…”