SLAM (signaling lymphocyte activation molecule)-associated protein (SAP) is a Src homology 2 (SH2) domain-containing adaptor expressed in T cells and natural killer cells. Its essential role inimmune responses is underscored by the recent finding that mutations in SAP result in a rare but fatal X-linked lymphoproliferative disease (XLP). Although SAP is known to associate with SLAM-family receptors, the exact molecular mechanism by which SAP regulates lymphocyte signaling remains elusive. We here report that in T cells, SAP associates with the PAK-interacting exchange factor (PIX), a guanine nucleotide exchange factor (GEF) specific for Rac͞Cdc42 GTPases. Moreover, SAP, PIX, and an activated form of Cdc42 form a complex in mammalian cells. We demonstrate that the SAP-PIX interaction is specific and is mediated by the C-terminal region of the SAP SH2 domain and the PIX SH3 domain. We further show that SAP is required for the recruitment of PIX to the SLAM-family receptors. Interestingly, overexpression of SAP, but not its homolog EAT-2, leads to a synergistic activation of nuclear factor of activating T cells (NFAT) in combination with a calcium signal in T cells. This SAP-mediated activation appears to be receptor-dependent and can be blocked by a dominant negative form of PIX. Taken together, our data strongly suggest that, in addition to the known SAP-interacting kinase Fyn, PIX may be another key player in SAP-mediated T cell activation.nuclear factor of activating T cells (NFAT) ͉ Cdc42 ͉ 2B4 ͉ Fyn X -linked lymphoproliferative disease (XLP) is an inherited severe or fatal immune dysfunction generally triggered by infection with Epstein-Barr virus (EBV) (1). XLP patients often develop a lethal fulminant infectious mononucleosis after EBV infection. Some patients also develop progressive dysgammaglobulinemia, malignant lymphomas, or autoimmunity such as vasculitis (2-4). Several immune cell types have been shown to exhibit functional alterations in XLP, including CD4 ϩ T cells, CD8 ϩ T cells, natural killer cells, and B cells (3). In 1998, three groups identified the gene aberrant in XLP, named as SAP͞ SH2D1A͞DSHP (5-7). The human SLAM (signaling lymphocyte activation molecule)-associated protein (SAP) is comprised of 128 amino acids, of which residues 6-102 contribute to an N-terminal SH2 domain. Many experiments have shown that the SH2 domain of SAP preferentially recognize specific tyrosinebased motifs containing TxYxxV͞I that are present within the cytoplasmic tails of SLAM and its related receptors, including 2B4, CD84, Ly9, NTB-A, and CRACC (1,8,9). Because XLP patients lack functional SAP, it has been speculated that the phenotypes of XLP likely reflect perturbed signaling downstream of one or more of these receptors, ultimately resulting in abnormal immune responses.Given the severe clinical manifestations in XLP patients, recent efforts have been focused on elucidating the signaling mechanism by which SAP regulates in T and natural killer cells. It has been shown that SAP recruits a Src kinase Fyn to...

Tumor necrosis factor alpha (TNFalpha) triggers a signaling pathway converging on the activation of NF-kappaB, which forms the basis for many physiological and pathological processes. In a kinase gene screen using a NF-kappaB reporter, we observed that overexpression of casein kinase 1alpha (CK1alpha) enhanced TNFalpha-induced NF-kappaB activation, and a CK1alpha kinase dead mutant, CK1alpha (K46A), reduced NF-kappaB activation induced by TNFalpha. We subsequently demonstrated that CK1alpha interacted with receptor interacting protein 1 (RIP1) but not with TRADD, TRAF2, MEKK3, IKKalpha, IKKbeta, or IKKgamma in mammalian cells. RIP1 is an indispensable molecule in TNFalpha/NF-kappaB signaling. We demonstrated that CK1alpha interacted with and phosphorylated RIP1 at the intermediate domain. Finally, we showed that CK1alpha enhanced RIP1-mediated NF-kappaB activation. Taken together, our studies suggest that CK1alpha is another kinase that regulates RIP1 function in NF-kappaB activation.