p21 Ras/Impedes Mitogenic Signal Propagation Regulates Cytokine Production and Migration in CD4 T Cells

Abstract

The propensity of T cells to generate coordinated cytokine responses is critical for the host to develop resistance to pathogens while maintaining the state of immunotolerance to selfantigens. The exact mechanisms responsible for preventing the overproduction of proinflammatory cytokines including interferon (IFN)-␥ are not fully understood, however. In this study, we examined the role of a recently described Ras GTPase effector and repressor of the Raf/MEK/ERK cascade called impedes mitogenic signal propagation (Imp) in limiting the induction of T-cell cytokines. We found that stimulation of the T cell receptor complex leads to the rapid development of a physical association between Ras and Imp. Consistent with the hypothesis that Imp inhibits signal transduction, we also found that disengagement of this molecule by the Ras V12G37 effector loop mutant or RNA interference markedly enhances the activation of the NFAT transcription factor and IFN-␥ secretion. A strong output of IFN-␥ is responsible for the distinct lymphocyte traffic pattern observed in vivo because the transgenic or retroviral expression of Ras V12G37 caused T cells to accumulate preferentially in the lymph nodes and delayed their escape from the lymphoid tissue, respectively. Together, our results describe a hitherto unrecognized negative regulatory role for Imp in the production of IFN-␥ in T cells and point to Ras-Imp binding as an attractive target for therapeutic interventions in conditions involving the production of this inflammatory cytokine.The small GTPase Ras is a potent signaling molecule that can bind with numerous downstream effector molecules including the protein kinase Raf (1, 2). Raf in turn activates the mitogenactivated protein kinase (MAPK) 2 kinase (MEK)/extracellular signal-regulated kinase (Erk) cascade (3, 4). This signaling pathway controls many pivotal functions in T cells. The ERK kinases overcome SHP-1 phosphatase blockade of proximal T cell receptor (TCR) signaling (5), and more globally, influence the maturation of T cells in the thymus (6 -9) and production of interleukin-2 (IL-2) in the post-thymic peripheral T cells (10 -14). The observation that a relative lack of Ras/ERK signal is associated with inability of T cells to respond to an antigen further underscores the fundamental role of this signaling pathway in T cell stimulation (15-17). It is not surprising to find therefore that T cells and other cell types have developed inhibitors to control the magnitude and duration of the ERK signaling. Among the most prominent endogenous repressors of the Ras/ERK signaling are GTPase-activating proteins (18, 19), downstream of tyrosine kinase (Dok) adaptor proteins (20 -22), members of the Sprouty protein family (23, 24), diacylglycerol kinases (DGK-and DGK-␣) (25, 26), ERK-specific dual specificity phosphatases (27), and a molecule with an E3 ubiquitin ligase activity described recently as impedes mitogenic signal propagation (Imp) (28).Imp, also known as BRCA1-associated protein (BRAP2), was originally identified as a...

Signaling Threshold Regulation by the Ras Effector IMP

“…Hypomorphic studies (described below) have shown that this is not simply an artifact of overexpression. Transcriptional activation through ERK is likewise inhibited, as shown in reporter assays with Elk-1 (26), c-Fos (6), and NF-AT (26). The inhibitory function of IMP appears to be specific for the ERK pathway as other kinases such as JNK (c-Jun N-terminal kinase) and Akt are not affected (6).…”

“…Furthermore, immunofluorescence analysis revealed that IMP recruits KSR to distinct subcellular compartments, which is reversed upon Ras activation (6). Interestingly, IMP that has been modified with a CAAX motif (and is thus localized to the cell membrane) is an even more effective ERK pathway inhibitor than unmodified IMP; the CAAX effect is further enhanced in the IMP(C264A) mutant (26). This may suggest that the site of action of IMP is near the membrane, which is consistent with its roles in suppressing membrane localization of KSR1 complexes and as a Ras effector.…”

“…Similarly, IMP-depleted PC12 cells are sensitized to nerve growth factor such that neurite outgrowth is induced at normally subthreshold concentrations (6). Finally, inhibition of IMP expression has been shown to significantly increase cytokine production by human Jurkat T-cells and primary mouse CD4 T-cells upon engagement of the T-cell receptor complex (26). Thus, IMP functions as a signal threshold regulator for the ERK pathway by imposing a stimulus-responsive inhibitory mechanism that must itself be inhibited for signal transduction to occur.…”

“…The magnitude of ERK activation is also responsible for various outcomes in the selection and specification of certain T-cell populations (35,36). Depletion of IMP protein does not affect base-line activation of MEK and ERK (6) but rather elevates their response to mitogenic stimulation and cross-linking of the T-cell receptor complex (26). Similarly, IMP-depleted PC12 cells are sensitized to nerve growth factor such that neurite outgrowth is induced at normally subthreshold concentrations (6).…”

BRAP Activates Inflammatory Cascades and Increases the Risk for Carotid Atherosclerosis

“…BRAP is also known to impede mitogenic signal propagation (IMP), the threshold modulator of the Raf-mitogen-activated protein kinase-extracellular signal regulated kinase (Raf-MEK-ERK) signal pathway (12). By repressing the Raf-MEK-ERK signal cascades, BRAP influences the secretion of interferon-γ in CD4 T cells (13). However, its role in the cardiovascular system remains to be explored.…”