unc-73 is required for cell migrations and axon guidance in C. elegans and encodes overlapping isoforms of 283 and 189 kDa that are closely related to the vertebrate Trio and Kalirin proteins, respectively. UNC-73A contains, in order, eight spectrin-like repeats, a Dbl/Pleckstrin homology (DH/PH) element, an SH3-like domain, a second DH/PH element, an immunoglobulin domain, and a fibronectin type III domain. UNC-73B terminates just downstream of the SH3-like domain. The first DH/PH element specifically activates the Rac GTPase in vitro and stimulates actin polymerization when expressed in Rat2 cells. Both functions are eliminated by introducing the S1216F mutation of unc-73(rh40) into this DH domain. Our results suggest that UNC-73 acts cell autonomously in a protein complex to regulate actin dynamics during cell and growth cone migrations.

Induction of T cell antigen receptor (TCR) endocytosis has a significant impact on TCR signaling and T cell behavior, but the molecular interactions coordinating internalization of the activated TCR are poorly understood. Previously we have shown that TCR endocytosis is regulated by the Wiskott Aldrich Syndrome protein (WASp), a cytosolic effector which, upon interaction with the cdc42 Rho GTPase, couples TCR engagement to Arp 2/3 complex-mediated actin polymerization. Here we report that WASp associates in T cells with intersectin 2, an endocytic adaptor containing multiple domains including a Dbl homology (DH) domain with the potential to activate Rho GTPases. Intersectin 2 association with WASp increases after TCR engagement, and its overexpression in Cos-7 cells induces WASp translocation to endocytic vesicles within which intersectin 2 colocalizes with both WASp and cdc42. Intersectin 2, but not a DH domain-deleted (ΔDH) form of intersectin 2, and stimulation via the TCR also trigger the activation of cdc42. Induction of TCR internalization is also augmented by intersectin 2 and severely impaired by latrunculin B treatment. Thus, intersection 2 appears to function cooperatively with WASp and cdc42 to link the clathrin endocytic machinery to WASp-mediated actin polymerization and ultimately to occupancy-induced TCR endocytosis.

Guanine nucleotide exchange factors in the Dbl family activate Rho GTPases by accelerating dissociation of bound GDP, promoting acquisition of the GTP-bound state. Dbl proteins possess a approximately 200 residue catalytic Dbl-homology (DH) domain, that is arranged in tandem with a C-terminal pleckstrin homology (PH) domain in nearly all cases. Here we report the solution structure of the DH domain of human PAK-interacting exchange protein (betaPIX). The domain is composed of 11 alpha-helices that form a flattened, elongated bundle. The structure explains a large body of mutagenesis data, which, along with sequence comparisons, identify the GTPase interaction site as a surface formed by three conserved helices near the center of one face of the domain. Proximity of the site to the DH C-terminus suggests a means by which PH-ligand interactions may be coupled to DH-GTPase interactions to regulate signaling through the Dbl proteins in vivo.

Cellular damage caused by reactive oxygen species is believed to be a major contributor to age-associated diseases. Previously, we characterized the Brap2 ortholog (BRAP-2) and found that it is required to prevent larval arrest in response to elevated levels of oxidative stress. Here, we report that mutants display increased expression of SKN-1-dependent, phase II detoxification enzymes that is dependent on PMK-1 (a p38 MAPK ortholog). An RNA-interference screen was conducted using a transcription factor library to identify genes required for increased expression of the SKN-1 target in mutants. We identified ELT-3, a member of the GATA transcription factor family, as a positive regulator of:: expression. We found that ELT-3 interacts with SKN-1 to activate transcription and that is required for enhanced expression in the mutant Furthermore, nematodes overexpressing SKN-1 required ELT-3 for life-span extension. Taken together, these results suggest a model where BRAP-2 acts as negative regulator of SKN-1 through inhibition of p38 MAPK activity, and that the GATA transcription factor ELT-3 is required along with SKN-1 for the phase II detoxification response in .

IntroductionMast cells are multifunctional hematopoietic cells, important for both innate and specific immunity. 1 Immature mast cells leave the bone marrow to migrate to target tissues, mostly mucosal and connective tissues, where they undergo terminal differentiation and perform their biologic functions. 2 Stem cell factor (SCF) and its receptor Kit are essential for mast cell development in vivo, as shown by the phenotype of mice with null mutations in the Kit (White Spotting or W) or SCF (Steel or Sl) loci. Indeed, these mice lack tissue mast cells and also exhibit defects in hematopoiesis, pigmentation, and reproduction. 3 In bone marrow mast cells (BMMCs), activation of Kit, a type III receptor tyrosine kinase (RTK), stimulates cellular responses such as proliferation, survival, differentiation, chemotaxis, cell adhesion, and degranulation. 4 Upon SCF binding, the Kit receptor dimerizes, autophosphorylates, and subsequently transphosphorylates specific tyrosines (Y). The resulting phosphotyrosine (pY) residues serve as docking sites for Src homology 2 (SH2) domain-containing proteins, which control intracellular signaling pathways such as all 3 mitogen-activated protein kinases (MAPKs; ERK, p38, and c-jun N-terminal kinase [JNK]), phosphatidylinositol 3-kinase (PI-3K), and Janus kinases (JAK)/signal transducers and activators of transcription (STAT) pathways. 4,5 Recruitment of particular targets is mediated by the ability of SH2 domains to recognize specific pY-containing motifs on the activated receptor. Analyses of individual docking sites on Kit have provided valuable information about the activation of pathways emanating from this receptor. 6 Juxtamembrane Y567 and Y569 are critical recruitment sites for different regulatory signaling molecules. These include activators such as Src family kinases (Fyn and Lyn), Shp-2 phosphatase and Shc adaptor protein, or negative modulators such as Shp-1 phosphatase, Csk-homology kinase (Chk), suppressors of cytokine signaling (SOCS) proteins, and APS adaptor protein. 5,7,8 Mutational and "knock-in" studies on Kit Y567/Y569 have revealed their important role in cell development, proliferation, survival, and migration. 6,9-11 However, the biologic significance of these interactions is still poorly understood.Lnk is a member of the adaptor protein family that includes APS and SH2-B. All 3 members share common protein-protein interaction domains and motifs: a dimerization domain at the aminoterminus, a pleckstrin homology (PH) domain, an SH2 domain, and a conserved tyrosine near the carboxy-terminus. 12 Mice deficient for members of this family have demonstrated the positive (SH2-B) and negative (Lnk and APS) role of these adaptors in growth factor, cytokine, and antigen-receptor signaling. [13][14][15][16][17][18] In particular, Lnk nullizygous mice show mainly a profound perturbation in hematopoiesis. These animals exhibit splenomegaly together with fibrosis, expansion of hematopoietic stem cells (HSCs), and myeloid and B lymphoid progenitors. [17][18][19] Recent ana...

mSos1 has been implicated in coupling mammalian tyrosine kinases to the Ras GTPase. Because activation of Ras induced by growth factor stimulation likely requires the localization of mSos1 to the plasma membrane, we have investigated the possibility that the PH domain of mSos1 might mediate an interaction of mSos1 with phospholipid membranes. A glutathione S-transferase fusion protein containing the pleckstrin homology (PH) domain of mSos1 bound specifically and tightly to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) with a K d of 1.8 ؎ 0.4 M. This interaction was saturable and was competed away with the soluble head group of PI(4,5)P 2 , inositol 1,4,5-triphosphate. Substitution of Arg 452 within the PH domain with Ala had only a slight effect on binding to PI(4,5)P 2 , whereas substitution of Arg 459 severely compromised the ability of the mSos1 PH domain to bind to PI(4,5)P 2 containing vesicles. Purified full-length mSos1 and mSos1 complexed with Grb2 were also tested for binding to various phosphoinositol derivatives and demonstrated a specific interaction with PI(4,5)P 2 , although these interactions were weaker (K d ‫؍‬ ϳ53 and ϳ69 M, respectively) than that of the PH domain alone. These findings suggest that the PH domain of mSos1 can interact in vitro with phospholipid vesicles containing PI(4,5)P 2 and that this interaction is facilitated by the ionic interaction of Arg 459 with the negatively charged head group of PI(4,5)P 2 . The association of the mSos1 PH domain with phospholipid may therefore play a role in regulating the function of this enzyme in vivo.

Aldose reductase (ALR2) shows a strong specificity for its nucleotide coenzyme, binding NADPH much more tightly than NADH (KD of < 1 microM versus 1.2 mM respectively). Interactions responsible for this specificity include salt linkages between the highly conserved residues Lys-262 and Arg-268, and the 2'-phosphate of NADP(H). Previous studies show that mutation of Lys-262 results in an increase in the Km for both coenzyme and aldehyde substrate, as well as in the kcat of reduction. The present study shows that mutation of Arg-268 to methionine results in a 36-fold increase in Km and 205-fold increase in KD for NADPH, but little change in Km for DL-glyceraldehyde or in the kcat of the reaction. Calculation of free energy changes show that the 2'-phosphate of NADPH contributes 4.7 kcal/mol of binding energy to its interaction with WT-hALR2. For the R268M mutant, the interaction of NADPH was destabilized by 3.2 kcal/mol, indicating that the mutation decreases the binding energy of NADPH by 65%. The effect of removing Arg-268 in the absence of the 2'-phosphate of NADPH was virtually identical to the destabilization of the activation energy in the absence of the 2'-phosphate itself (1.9 versus 2.0 kcal/mol, respectively). Therefore, while the 2'-phosphate of the coenzyme plays a role in both coenzyme binding and transition state stabilization during catalysis, the role of Arg-268 lies strictly in tighter coenzyme binding.

No abstract