The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. We report two previously unrecognized actions of NIK: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressing a kinase-inactive NIK, suppressed for NIK expression with siRNA oligonucleotides, or expressing ezrin T567A that cannot be phosphorylated. These data suggest that direct phosphorylation of ERM proteins by NIK constitutes a signaling mechanism controlling growth factor-induced membrane protrusion and cell morphology.ezrin ͉ moesin ͉ ste20 kinase ͉ membrane protrusion T he Nck-interacting kinase (NIK) is a member of the germinal center kinase subfamily of Ste20͞MAP4K serine͞threonine kinases (1). Closely related to NIK are the mammalian kinases TNIK (2), MINK (3), and NRK͞NESK (4) and orthologs Misshapen (Msn) in Drosophila (5) and MIG-15 in Caenorhabditis elegans (6). NIK and its orthologs share a common function in regulating cell shape and migration. In mice, homozygous knockout of NIK results in early embryonic lethality with defects in mesoderm migration (7), and expression of kinase-inactive NIK attenuates epithelial cell invasion (8). Msn functions in determining epithelial polarity, dorsal closure. and neuronal targeting (5, 9, 10), and MIG-15 controls axonal navigation (6). NIK (1) and Msn (5) also share a conserved activation of the JNK pathway. NIK, however, does not directly phosphorylate JNK, nor do NIK nullizygous embryos precisely phenocopy mice lacking JNK1 or JNK2 (7). Additionally, activation of JNK is not associated with dynamic changes in cell morphology. Hence, NIK substrates that control cell morphogenesis have not been identified.We now report that the ERM proteins ezrin, radixin, and moesin are substrates for NIK. ERM proteins regulate cell morphology by cross-linking actin filaments to the plasma membrane. The N-terminal FERM (4.1 ERM) domain of ERM proteins binds to integral plasma membrane proteins and the C terminus binds F-actin (11). ERM proteins control cell shape primarily by regulating membrane protrusions and cell-substrate adhesion. In epithelial cells ERM proteins are necessary for the formation of apical microvilli (12)(13)(14). In fibroblasts ERM proteins regulate the assembly of focal adhesions (...
Abstract. We report the complete sequence of Drosophila alpha-spectrin and show that it is similar to vertebrate nonerythroid spectrins. As in vertebrates, the alpha subunit consists of two large domains of repetitive sequence (segments 1-9 and 11-19) separated by a short nonrepetitive sequence (segment 10). The 106-residue repetitive segments are defined by a consensus sequence of 54 residues. Chicken alphaspectrin (Wasenius, V.-M., M. Saraste, E Salven, M.Eramaa, L. Holm, V.-P. Lehto. 1989. J. Cell Biol. 108:79-93) shares 50 of these consensus positions. Through comparison of spectrin and alpha-actinin sequences, we describe a second lineage of spectrin segments (20 and 21) that differs from the 106-residue segments by an 8-residue insertion and by lack of many of the consensus residues. We present a model of spectrin evolution in which the repetitive lineage of spectrin segments and the nonrepetitive lineage of segments found in spectrin and alpha-actinin arose by separate multiplication events. THE amino acid sequences of the erythroid alpha-and beta-spectrin subunits include ~18 tandem repeats of 106 amino acid segments that presumably account for the observed shape of the molecule (Speicher and Marchesi, 1984). The amino acid sequence has been confirmed by DNA sequence analysis (Wasenius et al., 1985;Birkenmeier et al., 1985) and sequence data for vertebrate nonerythroid spectrin cDNA has since been obtained. Partial sequences of nonerythroid spectrin from rat brain (Leto et al., 1988), human fibroblasts, frog and chicken brain (McMahon et al., 1987) and, most recently, the full length sequence of chicken brain alpha-spectrin (Wasenius et al., 1989) indicate that these molecules are also composed largely of 106 amino acid repetitive segments.We recently described the purification, cloning of cDNAs and immunofluorescence localization of spectrin in Drosophila (Dubreuil et al., 1987;Byers et al., 1987Byers et al., , 1989Pesacreta et al., 1989). Here we assemble complete cDNA sequence data for the alpha subunit of Drosophila spectrin.Our results, which show that this protein is strikingly similar in invertebrates and vertebrates, imply an important conservation of spectrin function. In addition, other studies have shown that alpha-spectfin-like sequences occur in alpha-actinin and dystrophin (Baron et al., 1987;Koenig et al., 1988; Davison et al., 1989). We have recently identified a region D. Bar-Zvi's present address is the Department of Biology, Ben-Gurion University, Beer-sheva, Israel.The Drosophila alpha-spectrin DNA sequence that appears in this paper is filed with the GenBank under the accession number M26400.of sequence similarity between Drosophila beta-spectrin and alpha-actinin ) that complements the region of similarity between alpha-spectrin and alpha-actinin. These observations are incorporated into a model of the structure and evolution of spectrin. Materials and Methods Isolation of cDNA Clones and Sequencing StrategyIsolation of cDNA clones 9 and 10 was previously described . Clones 9 a...
Endogenous electric fields are generated lateral to skin R. Rivkah Isseroff 1 wounds, with the cathodal pole of the field residing in the center of the Departments of 1 Dermatology, wound. These fields are thought to be an important mechanism in 2 Microbiology, and 3 Molecular and Cellular guiding the migration of keratinocytes and other cells into wounds to Biology, University of California, Davis, CA effect healing. In this work, human dermal fibroblasts were exposed to direct current electric fields of physiological strength, and their migrational behavior was quantitated. Only random migration of human dermal fibroblasts was observed in direct-current electric fields under conditions that support the directional migration of human epidermal keratinocytes. Additionally, neither the presence of serum nor serum plus suggest that dermal fibroblasts do not respond to the endogenous electric
Phospholipase C gamma-catalyzed inositol phospholipid hydrolysis, a critical step in B cell antigen receptor signaling leading to second messenger generation and proliferation, depends upon tyrosine kinase activation. The B cell antigen receptor-associated tyrosine kinases p53/56lyn, p59fyn, p55blk, and p72syk are assumed to participate in receptor-initiated signaling. It is unknown, however, which of these kinases is involved in the tyrosine phosphorylation and resulting activation of phospholipase C gamma in response to antigen receptor cross-linking. We have used a fusion protein containing the tandem src homology-2 (SH2) domains of phospholipase C gamma 1 (PLC gamma 1) to identify B cell kinases which associate with PLC gamma 1. Using an in vitro kinase assay, we demonstrate SH2-dependent association of tyrosine kinase activity from anti-mu-stimulated B cells. The PLC gamma 1 SH2 domains associate with a prominent 70-72-kDa tyrosine phosphoprotein from anti-mu-stimulated, but not resting, B cells. Immunoblotting and secondary immunoprecipitation studies definitively identify this protein as p72syk. These results imply a physical interaction between PLC gamma 1 and p72syk in antigen receptor-stimulated B cells. This conclusion is confirmed by our ability to co-immunoprecipitate p72syk and PLC gamma 1 from lysates of anti-mu-stimulated B cells. These results implicate p72syk in the activation of phospholipase C gamma 1 during B cell antigen receptor signaling.
The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscopic (EM) levels in interleukin-2 (IL2)-stimulated cloned T-cells and concanavalin A-stimulated splenocytes. This translocation occurs 2-10 h after IL2 stimulation, and is reversed by the addition of anti-PRL antiserum into the extracellular culture medium. The nuclear localization of PRL in IL2 stimulated T-cells was confirmed by postembedding immunogold EM. The nuclear uptake of PRL after IL2 stimulation was further documented by EM studies using PRL-colloidal gold conjugates. These studies suggest that the intranuclear PRL is translocated from the extracellular medium via an endosomal/lysosomal pathway over a period of several hours. Finally, the requirement for PRL no later than 6 h after IL2 stimulation is demonstrated through the reversible inhibition of T-cell growth with anti-PRL antiserum.
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