Interaction between a Domain of the Negative Regulator of the Ras-ERK Pathway, SPRED1 Protein, and the GTPase-activating Protein-related Domain of Neurofibromin Is Implicated in Legius Syndrome and Neurofibromatosis Type 1.

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120(GAP). Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.

Spred1, a Suppressor of the Ras-ERK Pathway, Negatively Regulates Expansion and Function of Group 2 Innate Lymphoid Cells.

Cytokines from group 2 innate lymphoid cells (ILC2s) have been implicated in acute allergic responses, such as papain-induced lung inflammation. However, the means of homeostatic regulation of ILC2s have not been established. In this study, we demonstrated that Spred1, a negative regulator of the Ras-ERK pathway, plays an important role in the proliferation and apoptosis of ILC2s and in cytokine secretion from ILC2s. Intranasal administration of papain stimulated IL-5 and IL-13 production in the lung, which was enhanced when Spred1 was deleted. In vitro, Spred1(-/-) ILC2s proliferated faster than wild type ILC2s did and produced higher levels of cytokines in response to IL-33. On the contrary, a MEK inhibitor suppressed ILC2 proliferation and cytokine production. Spred1 deficiency resulted in stabilization of GATA3, which has been shown to play essential roles in the maintenance and cytokine production of ILC2. These data suggest that Spred1 negatively regulates ILC2 development and functions through the suppression of the Ras-ERK pathway.

TAK1-JNK axis mediates survival signal through Mcl1 stabilization in activated T cells.

TAK1, a member of MAPK kinase kinase (MAPKK-K) family, can activate JNK, p38 MAPK, and NF-κB signaling pathways. Although targeted gene disruption studies have demonstrated that TAK1 plays a critical role in T cell functions, precise functions of downstream mediators remain elusive. We used the chemical compound LL-Z1640-2, which preferentially suppressed MAPK activation but not NF-κB signal downstream of TAK1. LL-Z1640-2 blocked TCR-induced T cell proliferation and activation, confirming that a TAK1-mediated MAPK signal is essential for T cell activation. LL-Z1640-2 induced apoptosis of activated mouse splenic T cells in a caspase- and caspase-activated DNase-dependent manner. TAK1-JNK pathway, which is activated downstream of IL-2R, induced the phosphorylation of antiapoptotic protein Mcl1 in activated T cells, resulting in the stabilization of Mcl1 protein. Our data uncover that among signal transduction pathways downstream of TAK1, JNK mediates a survival program through Mcl1 stabilization downstream of IL-2R in activated T cells and that blockade of TAK1-JNK pathway can eliminate activated T cells by apoptosis.

Critical role of class IA PI3K for c-Rel expression in B lymphocytes.

The fact that the Xid mutation of Btk impairs the ability of pleckstrin homology domain of Btk to bind phosphatidylinositol-(3,4,5)-trisphosphate, a product of class IA phosphoinositide-3 kinases (PI3Ks), has been considered strong evidence for the hypothesis that Btk functions downstream of PI3Ks. We demonstrate here that the Xid mutation renders the Btk protein unstable. Furthermore, class IA PI3K- and Btk-deficient mice show different phenotypes in B-cell development, collectively indicating that PI3Ks and Btk differentially function in BCR signal transduction. Nevertheless, both PI3K and Btk are required for the activation of NF-kappaB, a critical transcription factor family for B-cell development and function. We demonstrate that PI3Ks maintain the expression of NF-kappaB proteins, whereas Btk is known to be essential for IkappaB degradation and the translocation of NF-kappaB to the nucleus. The loss of PI3K activity results in marked reduction of c-Rel and to a lesser extent RelA expression. The lentivirus-mediated introduction of c-Rel corrects both developmental and proliferative defects in response to BCR stimulation in class IA PI3K-deficient B cells. These results show that the PI3K-mediated control of c-Rel expression is essential for B-cell functions.

Negative feedback loop in T-cell activation through MAPK-catalyzed threonine phosphorylation of LAT.

Mitogen-activated protein kinase (MAPK) cascades are involved in a variety of cellular responses including proliferation, differentiation, and apoptosis. We have developed an expression screening method to detect in vivo substrates of MAPKs in mammalian cells, and identified a membrane protein, linker for activation of T cells (LAT), as an MAPK target. LAT, an adapter protein essential for T-cell signaling, is phosphorylated at its Thr 155 by ERK in response to T-cell receptor stimulation. Thr 155 phosphorylation reduces the ability of LAT to recruit PLCgamma1 and SLP76, leading to attenuation of subsequent downstream events such as [Ca2+]i mobilization and activation of the ERK pathway. Our data reveal a new role for MAPKs in a negative feedback loop in T-cell activation via threonine phosphorylation of LAT.