Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins ␣ 3 ␤ 1 and ␣ 6 ␤ 4 , have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both ␣ 3 ␤ 1 and ␣ 6 ␤ 4 mediate adhesion to laminin-5, only ␣ 3 ␤ 1 -dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced ζ-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.

We have previously reported the Ras‐dependent activation of the mitogen‐activated protein kinases p44 and p42, also termed extracellular signal‐regulated kinases (ERK)1 and 2 (ERK1/2), mediated through Gs‐coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas Gi‐ and Gq‐coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras‐GRF1 (CDC25Mm) by binding of Ca2+/calmodulin to its N‐terminal IQ domain, the mechanism of Ras activation through Gs‐coupled receptors is not fully understood. We report the endogenous expression of Ras‐GRF1 in HEK293 cells. Serotonin stimulation of HEK293 cells transiently expressing Gs‐coupled 5‐HT7 receptors induced protein kinase A‐dependent phosphorylation of the endogenous human Ras‐GRF1 on Ser927 and of transfected mouse Ras‐GRF1 on Ser916. Ras‐GRF1 overexpression increased basal and serotonin‐stimulated ERK1/2 phosphorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking (Ser916Asp/Glu) phosphorylation did not alter these effects. However, the deletion of amino acids 1–225, including the Ca2+/calmodulin‐binding IQ domain, from Ras‐GRF1 reduced both basal and serotonin‐stimulated ERK1/2 phosphorylation. Furthermore, serotonin treatment of HEK293 cells stably expressing 5‐HT7 receptors increased [Ca2+]i, and the serotonin‐induced ERK1/2 phosphorylation was Ca2+‐dependent. Therefore, both cAMP and Ca2+ may contribute to the Ras‐dependent ERK1/2 activation after 5‐HT7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras.

Here, we examined the functional involvement of heterotrimeric G-proteins in TCR-induced immune responses. TCR/CD3 crosslinking resulted in activation of both Gaq and Gas, but not Gai-2. Targeting of Gas, Gai-2 and Gaq using siRNA demonstrated a specific role of Gaq in TCR signaling. Jurkat TAg T cells with Gaq knockdown displayed reduced activation of Lck and LAT phosphorylation, but paradoxically showed sustained ERK1/2 phosphorylation and increased NFAT-AP-1-reporter activity implicating Gaq in the negative control of downstream signaling and IL-2-promoter activity. Primary T cells isolated from Gaq-deficient mice had a similar TCR signaling response with reduced proximal LAT phosphorylation, sustained ERK1/2 phosphorylation and augmented immune responses including increased secretion of IL-2, IL-5, IL-12 and TNF-a. The effects on NFAT-AP-1-reporter activity were sensitive to the Src family kinase inhibitor PP2 and were reversed by transient expression of constitutively active Lck. Furthermore, expression of constitutively active Gaq Q209L elevated Lck activity and Zap-70 phosphorylation. Together these data argue for a role of Gaq in the fine-tuning of proximal TCR signals at the level of Lck and a negative regulatory role of Gaq in transcriptional activation of cytokine responses. Key words: Signal transduction . T cells . TCR . Transgenic/knockout mice IntroductionThe TCR/CD3 complex is composed of a ligand-binding TCR-ab heterodimer and signal transducing dimers of CD3eg, CD3ed and zz that upon interaction with its MHC-peptide ligand initiate an activation process that, although not fully understood, probably involves both receptor clustering and conformational changes of the cytoplasmic part of the CD3 complex [1,2]. Considering the physiological significance of correct T-cell activation, this signal interpretation probably involves complex molecular integration proximal to the TCR. A key early event is the mobilization and activation of the protein tyrosine kinase Lck, which in turn phosphorylates ITAM within the CD3 component that can recruit ZAP-70 through its two SH2-domains [3]. ZAP-70 is then phosphorylated by Lck on Y493 leading to full catalytic activation [4]. One important substrate of activated ZAP-70 is the transmembrane adaptor LAT, which acts as a scaffold to assemble signaling complexes essential for T-cell activation [5].The 3208TCR necessary for the development and activation of T cells. In addition, several lines of evidence suggest that initiation of modulating pathways is controlled by Lck. Both TCR desensitization by internalization and induction of apoptosis through the mitochondrial death pathway are regulated by Lck [7,8]. Furthermore, the activation of Lck by partial antagonist and the ability of Lck to negatively regulate superantigen-induced T-cell activation support a dual role of Lck [9,10]. Interestingly, acute elimination of Lck in either Jurkat or primary T cells using RNA interference results in sustained and elevated ERK1/2 phosphorylation and augmented NFAT-reporter ac...

T cells with short interfering RNA-mediated Lck-knockdown (kd) display paradoxical hyper-responsiveness upon TCR ligation. We have previously reported a possible mechanism for T-cell activation in cells with low levels of Lck depending on Grb2-SOS1 recruitment to the zeta-chain of TCR/CD3 (Methi et al., Eur. J. Immunol. 2007, 37: 2539-2548. Here, we show that short interfering RNA-mediated targeting of Lck caused a dramatic reduction in c-Cbl phosphorylation and a general reduction in protein ubiquitination after TCR stimulation. Specifically, this resulted in reduced ubiquitination of the zeta-chain, yet internalization of TCR/CD3 appeared to be normal after receptor engagement. However, zeta-chain levels were elevated in Lck-kd cells, and confocal microscopy revealed reduced colocalization of CD3-containing vesicles with endosomal and lysosomal compartments.We hypothesize that prolonged stability of internalized T-cell receptor complex may result in extended signaling in T cells with low Lck levels. Key words: Cbl Á CD3 Á Lck Á T cells Á T-cell receptors IntroductionEngagement of the TCR leads not only to activation of T cells, but also to internalization and lysosomal degradation of the receptor complex [1]. The latter process serves to terminate signaling, and has been shown to be dependent on the tyrosine kinase activity of Lck [2,3] and ubiquitination by c-Cbl [4][5][6]. The E3 ubiquitin ligase c-Cbl functions as a negative regulator of many signaling pathways and ubiquitination marks active enzymes and receptors for degradation [reviewed in 7 and 8]. Notable targets for c-Cbl-mediated ubiquitination are Lck [9], Vav [10], Fyn [11][12][13], , as well as the already mentioned TCR/ CD3-complex. c-Cbl itself is activated by tyrosine phosphorylation on several residues, most importantly Y700, Y731 and Y774 [13,[15][16][17]. These residues are not, however, substrates of Lck or but of Fyn and Syk [13,16,18,19], which are activated directly or indirectly by Lck [20].Cbl exists in two main isoforms, c-Cbl and Cbl-b, with a high level of sequence conservation. Consistent with the negative regulation assigned to the Cbl family of proteins, T cells from c-Cbl À/À and Cbl-b À/À mice were hyperactive upon TCR engagement, although some biochemical distinctions between the phenotypes exist [21][22][23][24][25]. T cells from double-knockout mice lacking both c-Cbl and Cbl-b failed to modulate surface TCR after ligand engagement, resulting in sustained TCR signaling and ERK1/2 phosphorylation. However, signaling through the major TCR pathways were not increased [26]. These observations are comparable to what we found in T cells with low levels of Lck, which also display prolonged ERK1/2 activation while expression levels of other proteins (Fyn, Csk, PKCa, PLCg1, LAT, FAK, and Pyk2) remained unaffected [27,28]. We therefore investigated the impact of short interfering RNA (siRNA)-mediated Lck-knockdown (kd) on c-Cbl activity and TCR/CD3 turnover in T cells. As expected, c-Cbl phosphorylation and ubiquitination of the z-c...