Deborah Yablonski

Identification of a Phospholipase C-γ1 (PLC-γ1) SH3 Domain-Binding Site in SLP-76 Required for T-Cell Receptor-Mediated Activation of PLC-γ1 and NFAT

ABSTRACT SLP-76 is an adapter protein required for T-cell receptor (TCR) signaling. In particular, TCR-induced tyrosine phosphorylation and activation of phospholipase C-γ1 (PLC-γ1), and the resultant TCR-inducible gene expression, depend on SLP-76. Nonetheless, the mechanisms by which SLP-76 mediates PLC-γ1 activation are not well understood. We now demonstrate that SLP-76 directly interacts with the Src homology 3 (SH3) domain of PLC-γ1. Structure-function analysis of SLP-76 revealed that each of the previously defined protein-protein interaction domains can be individually deleted without completely disrupting SLP-76 function. Additional deletion mutations revealed a new, 67-amino-acid functional domain within the proline-rich region of SLP-76, which we have termed the P-1 domain. The P-1 domain mediates a constitutive interaction of SLP-76 with the SH3 domain of PLC-γ1 and is required for TCR-mediated activation of Erk, PLC-γ1, and NFAT (nuclear factor of activated T cells). The adjacent Gads-binding domain of SLP-76, also within the proline-rich region, mediates inducible recruitment of SLP-76 to a PLC-γ1-containing complex via the recruitment of both PLC-γ1 and Gads to another cell-type-specific adapter, LAT. Thus, TCR-induced activation of PLC-γ1 entails the binding of PLC-γ1 to both LAT and SLP-76, a finding that may underlie the requirement for both LAT and SLP-76 to mediate the optimal activation of PLC-γ1.

A NCK-PAK1 SIGNALING MODULE IS REQUIRED FOR T-CELL RECEPTOR-MEDIATED ACTIVATION OF NFAT, BUT NOT OF JNK

The T‐cell antigen receptor (TCR) triggers a signaling cascade initiated by the tyrosine kinase Lck and requiring the proto‐oncogene p95vav. Vav is activated by Lck and can function as a guanine nucleotide exchange factor for the Rho‐family GTPases, Rac1 and Cdc42. To investigate the involvement of these GTPases in TCR signaling, we focused on their well characterized effector, Pak1. This serine/threonine kinase is activated by GTP‐bound Rac1 or Cdc42. However, its role in mediating downstream signaling events is controversial. We observed rapid, TCR‐dependent activation of Pak1 and TCR‐inducible association of Pak1 with Nck, which was tyrosine phosphorylated following stimulation. Pak1 activation occurred independently of Ras activation or calcium flux, but was dependent on the Lck tyrosine kinase, and was downstream of Vav and Cdc42. Dominant negative Pak1 or Nck specifically inhibited TCR‐mediated activation of the nuclear factor of activated T cells (NFAT) transcription factor. TCR‐mediated activation of Erk2 was also inhibited by dominant negative Pak. However, Pak1 activation was neither necessary nor sufficient for TCR‐dependent c‐Jun N‐terminal kinase (JNK) activation. Therefore, Pak1 acts downstream of Vav and is required for activation of Erk2 and NFAT by a JNK‐independent pathway. This is the first demonstration of a requirement for Pak to mediate the regulation of gene expression by an extracellular ligand.

Linker for Activation of T Cells, ζ-Associated Protein-70, and Src Homology 2 Domain-Containing Leukocyte Protein-76 are Required for TCR-Induced Microtubule-Organizing Center Polarization

Engagement of the T cell with Ag on an APC results in a series of immediate signaling events emanating from the stimulation of the TCR. These events include the induced phosphorylation of a number of cellular proteins with a subsequent increase in intracellular calcium and the restructuring of the microtubule and actin cytoskeleton within the T cell. This restructuring of the cytoskeleton culminates in the polarization of the T cell’s secretory apparatus toward the engaging APC, allowing the T cell to direct secretion of cytokines toward the appropriate APC. This polarization can be monitored by analyzing the position of the microtubule-organizing center (MTOC), as it moves toward the interface of the T cell and APC. The requirements for MTOC polarization were examined at a single-cell level by studying the interaction of a Jurkat cell line expressing a fluorescently labeled MTOC with Staphylococcal enterotoxin superantigen-bound Raji B cell line, which served as the APC. We found that repolarization of the MTOC substantially followed fluxes in calcium. We also used immobilized anti-TCR mAb and Jurkat signaling mutants, defective in TCR-induced calcium increases, to determine whether signaling components that are necessary for a calcium response also play a role in MTOC polarization. We found that ζ-associated protein-70 as well as its substrate adaptor proteins linker for activation of T cells and Src homology 2 domain-containing leukocyte protein-76 are required for MTOC polarization. Moreover, our studies revealed that a calcium-dependent event not requiring calcineurin or calcium/calmodulin-dependent kinase is required for TCR-induced polarization of the MTOC.

A PAK1-PIX-PKL complex is activated by the T-cell receptor independent of Nck, Slp-76 and LAT

Given the importance of the Rho GTPase family member Rac1 and the Rac1/Cdc42 effector PAK1 in T‐cell activation, we investigated the requirements for their activation by the T‐cell receptor (TCR). Rac1 and PAK1 activation required the tyrosine kinases ZAP‐70 and Syk, but not the cytoplasmic adaptor Slp‐76. Surprisingly, PAK1 was activated in the absence of the transmembrane adaptor LAT while Rac1 was not. However, efficient PAK1 activation required its binding sites for Rho GTPases and for PIX, a guanine nucleotide exchange factor for Rho GTPases. The overexpression of βPIX that either cannot bind PAK1 or lacks GEF function blocked PAK1 activation. These data suggest that a PAK1–PIX complex is recruited to appropriate sites for activation and that PIX is required for Rho family GTPase activation upstream of PAK1. Furthermore, we detected a stable trimolecular complex of PAK1, PIX and the paxillin kinase linker p95PKL. Taken together, these data show that PAK1 contained in this trimolecular complex is activated by a novel LAT‐ and Slp‐76‐independent pathway following TCR stimulation.

The Src Kinase p56 lck Up-regulates VLA-4 Integrin Affinity IMPLICATIONS FOR RAPID SPONTANEOUS AND CHEMOKINE-TRIGGERED T CELL ADHESION TO VCAM-1 AND FIBRONECTIN

In circulating lymphocytes, the VLA-4 integrin preexists in multiple affinity states that mediate spontaneous tethering, rolling, and arrest on its endothelial ligand, vascular cell adhesion molecule-1 (VCAM-1). The regulation and function of VLA-4 affinity in lymphocytes has never been elucidated. We show here that p56 lck , the major Src kinase in T cells, is a key regulator of high affinity VLA-4. This high affinity is essential for the rapid development of firm adhesion of resting T cells to VCAM-1 and to their extracellular matrix ligand, fibronectin. Lck-regulated VLA-4 function does not require intact TCR nor several key components of the TCR signaling pathway, including ZAP-70 and SLP-76. Furthermore, stimulation of p56 lck by the phosphatase inhibitor, pervanadate, triggers firm VLA-4-dependent adhesion to VCAM-1. Although Lck is not required for chemokine receptor signaling to mitogen-activated protein kinase, the presence of Lck-regulated high affinity VLA-4 also facilitates firm adhesion triggered by the chemokine, SDF-1, at short-lived contacts. Surprisingly, bond formation rates, ability to tether cells to VLA-4 ligand, and VLA-4 tether bond stability under shear flow are not affected by VLA-4 affinity or Lck activity. Thus, the ability of high affinity VLA-4 to arrest cells on VCAM-1 under flow arises from instantaneous post-ligand strengthening rather than from increased kinetic stability of individual VLA-4 bonds. These results suggest that p56 lck maintains high affinity VLA-4 on circulating lymphocytes, which determines their ability to strengthen VLA-4 adhesion and rapidly respond to proadhesive chemokine signals at endothelial sites.

SLP-76 mediates and maintains activation of the Tec family kinase ITK via the T cell antigen receptor-induced association between SLP-76 and ITK.

ITK (IL-2-inducible T cell kinase), a Tec family protein tyrosine kinase (PTK), is one of three PTKs required for T cell antigen receptor (TCR)-induced activation of phospholipase C-γ1 (PLC-γ1). Like Src and Abl family PTKs, ITK adopts an inactive, “closed” conformation, and its conversion to the active conformation is not well understood, nor have its direct substrates been identified. In a side-by-side comparison of ITK and ZAP-70 (ζ chain-associated protein kinase of 70 kDa), ITK efficiently phosphorylated Y783 and Y775 of PLC-γ1, two phosphorylation sites that are critical for its activation, whereas ZAP-70 did not. SLP-76 (SH2-domain-containing leukocyte protein of 76 kDa), an adaptor required for TCR-induced activation of PLC-γ1, was required for the phosphorylation of both PLC-γ1 sites in intact cells. Furthermore, this event depended on the N-terminal tyrosines of SLP-76. Likewise, SLP-76, particularly its N-terminal tyrosines, was required for TCR-induced tyrosine phosphorylation and activation of ITK but was not required for the phosphorylation or activation of ZAP-70. Both ZAP-70 and ITK phosphorylated SLP-76 in vitro; thus, both PTKs are potential regulators of SLP-76, but only ITK is regulated by SLP-76. Upon TCR stimulation, a small fraction of ITK bound to SLP-76. This fraction, however, encompassed most of the catalytically active ITK. Catalytic activity was lost upon mild elution of ITK from the SLP-76-nucleated complex but was restored upon reconstitution of the complex. We propose that SLP-76 is required for ITK activation; furthermore, an ongoing physical interaction between SLP-76 and ITK is required to maintain ITK in an active conformation.

Mechanisms of signaling by the hematopoietic-specific adaptor proteins, slp-76 and lat and their b cell counterpart, blnk/slp-65

Adaptor proteins lack catalytic activity and contain only protein-protein interaction domains. They have been shown to interact with an ever-growing number of signaling proteins and to play essential roles in many signaling pathways. SLP-76 and LAT are cell-type-specific adaptor proteins expressed in T cells, NK cells, platelets, and mast cells. In these cell types, SLP-76 and LAT are required for signaling by immunoreceptor tyrosine-based activation motif(ITAM)-containing receptors, including the T cell receptor (TCR), the pre-TCR, the high-affinity Fc epsilon receptor, and the platelet GPVI collagen receptor. In B cells, an analogous adaptor, BLNK/SLP-65, is required for signaling by the ITAM-containing B cell receptor. This review summarizes recent research on SLP-76, LAT, and BLNK. A major challenge in understanding adaptor protein function has been to sort out the many interactions mediated by adaptor proteins and to define the mechanisms by which adaptors mediate critical signaling events. In the case of LAT, SLP-76, and BLNK, the availability of tractable genetic systems, deficient in expression of each of these adaptor proteins, has facilitated in-depth investigation of their signaling functions and mechanisms of action. The picture that has emerged is one in which multiple adaptor proteins cooperate to bring about the formation of a large signaling complex, localized to specialized lipid microdomains within the cell membrane and known as GEMs. Adaptors not only recruit signaling proteins, but also play an active role in regulating the conformation and activation of many of the proteins recruited to the complex. In particular, recent research has shed light on the mechanisms by which multiple adaptor proteins cooperate to bring about the recruitment and activation of phospholipase C gamma in response to the activation of ITAM-containing receptors.

Autophosphorylation-dependent degradation of Pak1, triggered by the Rho-family GTPase, Chp

The Paks (p21-activated kinases) Pak1, Pak2 and Pak3 are among the most studied effectors of the Rho-family GTPases, Rac, Cdc42 (cell division cycle 42) and Chp (Cdc42 homologous protein). Pak kinases influence a variety of cellular functions, but the process of Pak down-regulation, following activation, is poorly understood. In the present study, we describe for the first time a negative-inhibitory loop generated by the small Rho-GTPases Cdc42 and Chp, resulting in Pak1 inhibition. Upon overexpression of Chp, we unexpectedly observed a T-cell migration phenotype consistent with Paks inhibition. In line with this observation, overexpression of either Chp or Cdc42 caused a marked reduction in the level of Pak1 protein in a number of different cell lines. Chp-induced degradation was accompanied by ubiquitination of Pak1, and was dependent on the proteasome. The susceptibility of Pak1 to Chp-induced degradation depended on its p21-binding domain, kinase activity and a number of Pak1 autophosphorylation sites, whereas the PIX- (Pak-interacting exchange factor) and Nck-binding sites were not required. Together, these results implicate Chp-induced kinase autophosphorylation in the degradation of Pak1. The N-terminal domain of Chp was found to be required for Chp-induced degradation, although not for Pak1 activation, suggesting that Chp provides a second function, distinct from kinase activation, to trigger Pak degradation. Collectively, our results demonstrate a novel mechanism of signal termination mediated by the Rho-family GTPases Chp and Cdc42, which results in ubiquitin-mediated degradation of one of their direct effectors, Pak1.

Dual Role of SLP-76 in Mediating T Cell Receptor-induced Activation of Phospholipase C-γ1

Phospholipase C-γ1 (PLC-γ1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-γ1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-γ1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-γ1 GEM recruitment, but were required for PLC-γ1 phosphorylation at Tyr783. Thus, GEM recruitment can be insufficient for full activation of PLC-γ1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-γ1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-γ1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-γ1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.

A Pak- and Pix-dependent branch of the SDF-1α signalling pathway mediates T cell chemotaxis across restrictive barriers

Pak (p21-activated kinase) serine/threonine kinases have been shown to mediate directional sensing of chemokine gradients. We hypothesized that Pak may also mediate chemokine-induced shape changes, to facilitate leucocyte chemotaxis through restrictive barriers, such as the extracellular matrix. A potent inhibitor, Pak(i), was characterized and used to probe the role of Pak-family kinases in SDF-1alpha (stromal-cell derived factor-1alpha/CXCL12)-induced chemotaxis in a T cell model. Pak(i) potently inhibited SDF-1alpha-induced Pak activation by a bivalent mechanism, as indicated by its complete inactivation upon point mutation of two binding sites, but partial inactivation upon mutation of either site alone. Importantly, Pak(i) was not toxic to cells over the time frame of our experiments, since it did not substantially affect cell surface expression of CXCR4 (CXC chemokine receptor 4) or integrins, cell cycle progression, or a number of ligand-induced responses. Pak(i) produced dose-dependent inhibition of SDF-1alpha-induced migration through rigid filters bearing small pores; but unexpectedly, did not substantially affect the magnitude or kinetics of chemotaxis through filters bearing larger pores. SDF-1alpha-induced Pak activation was partly dependent on PIX (Pak-interactive exchange factor); correspondingly, an allele of beta-PIX that cannot bind Pak inhibited SDF-1alpha-induced chemotaxis through small, but not large pores. By contrast, other key players in chemotaxis: G(i), PI3K (phosphoinositide 3-kinase), and the Rho-family G-proteins, Rac and Cdc42 (cell division cycle 42), were required for SDF-1alpha-induced migration regardless of the barrier pore-size. These studies have revealed a distinct branch of the SDF-1alpha signalling pathway, in which the Rac/Cdc42 effector, Pak, and its partner, PIX, specifically regulate the cellular events required for chemokine-induced migration through restrictive barriers.