A 58-kDa Shc protein is present in Xenopus eggs and is phosphorylated on tyrosine residues upon egg activation.

A 58-kDa protein was detected in Xenopus egg lysate by SDS-PAGE and immunoblotting with an antibody raised against adaptor protein Shc, a well known tyrosine kinase substrate in numerous biological events. Tyrosine phosphorylation of the Xenopus Shc protein (p58 xShc) was found to increase 2.3 +/- 0.4-fold (n = 3) upon fertilization. Pretreatment of eggs with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation. Tyrosine phosphorylation of p58 xShc was also observed when eggs were activated parthenogenetically by an integrin-interacting RGDS-peptide which is known to cause egg activation accompanied by intracellular calcium release. On the other hand, other egg-activating treatments such as electrical shock and calcium ionophore, which directly induce the elevation of intracellular calcium, did not show such an effect. It is also suggested that the phosphorylated p58 xShc may play a role unique to the egg activation process because we found that there was no increase of Shc-Grb2 complex after fertilization. These results demonstrate that p58 xShc is a substrate of egg tyrosine kinases which may be activated by sperm-egg interaction and suggest that the phosphorylated p58 xShc may act upstream of the calcium-dependent pathway of egg activation.     

Junctional adhesion molecule (JAM) is phosphorylated by protein kinase C upon platelet activation

Junctional adhesion molecule (JAM) is a member of the immunoglobulin superfamily (IgSF) expressed in tight junctions of epithelial cells and endothelial cells, and implicated in transendothelial migration of leukocytes. Recently, JAM is reported to be constitutively expressed on circulating monocytes, neutrophils, lymphocytes subsets, and platelets. However, the role of JAM is not known. Here, we examined how phosphorylaton of JAM is regulated upon platelet activation. Phosphorylation of JAM was induced by thrombin, collagen, but not by ADP. The phosphorylated amino acids were shown to be serine residues by phosphoamino acid analysis. Inhibition of JAM's phosphorylation by PKC inhibitors and Ca(++) chelator suggests the involvement of conventional types of PKCs. By in vitro kinase assays, we demonstrated that JAM could be directly phosphorylated by cPKCs. We also demonstrated phosphorylation of Ser 284, a putative PKC phosphorylation site, by immunoblotting with anti-phosphoserine-JAM antibody in thrombin-stimulated platelets. In addition to the phosphorylation, JAM seemed to form clusters at several sites of cell-cell contact in aggregated platelets by immunoelectron microscopic study. We speculate that JAM may be directly phosphorylated by cPKC(s)upon platelet activation and that the phosphorylationmight be involved in platelet activation.     

The GAGA protein of Drosophila is phosphorylated by CK2

The GAGA factor of Drosophila is a sequence-specific DNA-binding protein that contributes to multiple processes from the regulation of gene expression to the structural organisation of heterochromatin and chromatin remodelling. GAGA is known to interact with various other proteins (tramtrack, pipsqueak, batman and dSAP18) and protein complexes (PRC1, NURF and FACT). GAGA functions are likely regulated at the level of post-translational modifications. Little is known, however, about its actual pattern of modification. It was proposed that GAGA can be O-glycosylated. Here, we report that GAGA519 isoform is a phosphoprotein that is phosphorylated by CK2 at the region of the DNA-binding domain. Our results indicate that phosphorylation occurs at S388 and, to a lesser extent, at S378. These two residues are located in a region of the DNA-binding domain that makes no direct contact with DNA, being dispensable for sequence-specific recognition. Phosphorylation at these sites does not abolish DNA binding but reduces the affinity of the interaction. These results are discussed in the context of the various functions and interactions that GAGA supports.     

The T cell antigen receptor zeta chain is tyrosine phosphorylated upon activation

The T cell antigen receptor is composed of at least seven chains derived from six different gene products. Upon stimulation, several chains can be phosphorylated. Two of these, CD3-gamma and CD3-epsilon are phosphorylated on serine residues. In addition, a 21-kDa nonglycosylated receptor component is phosphorylated, upon activation, on tyrosine residues. We have referred to this phosphoprotein as p21 because we have previously not been able to assign the tyrosine phosphorylation to any of the described receptor subunits (Samelson, L. E., Patel, M. D., Weissman, A. M., Harford, J. B., and Klausner, R. D. (1986) Cell 46, 1083-1090). In this paper, we demonstrate that it is the 16-kDa zeta chain which is the tyrosine phosphorylated subunit, and thus the p21 nomenclature can be replaced. This phosphorylation results in a shift of the apparent Mr of zeta to 21 kDa. Proof that p21 is tyrosine phosphorylated zeta was afforded by a number of approaches. Specific anti-zeta antibodies directly precipitated phospho-p21. Metabolically labeled protein corresponding to p21 could only be observed after activation. When this 21-kDa band was isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment with alkaline phosphatase, its migration was identical with that of zeta. Furthermore, peptide mapping of metabolically labeled p21 (after gel isolation and dephosphorylation) showed it to be indistinguishable from p21. Thus, one of the early events of T cell activation is the tyrosine phosphorylation of the zeta chain of the T cell antigen receptor.     

Fire exit is a potential four transmembrane protein expressed in developing Drosophila glia

Glia from many diverse organisms play a number of important roles during the development of the nervous system. Therefore, knowing the molecules that control glial cell function will further our understanding of the mechanisms that control nervous system development. We have isolated a novel gene in Drosophila melanogaster that is expressed in a subset of the peripheral glia. We call this gene "Fire exit" (Fie), as the glia that express this gene do so during a time when they mark the entry and exit point of axons at the CNS/PNS boundary. This subset of peripheral glia act as intermediate targets during pathfinding and migration of the sensory axons in particular. Fire exit has been cloned and found to encode a novel transmembrane protein. Fire exit belongs to a group of proteins identified in the Drosophila melanogaster and Anopheles gambiae databases which contain four predicted transmembrane domains and a shared intracellular motif. Mutations that remove the fire exit protein have no obvious disruption to glial function. On the other hand, glia expressing the Fire exit gene bridge the transition zone between CNS and PNS and play a role in sensory axon guidance. Therefore, it appears that, while the glia that express this protein mediate axon guidance, Fire exit itself plays a nonessential part in this function. A role for Fire exit in glial development may be suggested by its evolutionary relationship to a family of lysosome-associated proteins called LAPTMs and suggests that Fire exit may function in intracellular transport during glial development.     

Drosophila melanogaster H1 histone is phosphorylated stably.

Phosphorylation of histone H1 is developmentally regulated in Drosophila spp. It cannot be detected in preblastoderm embryos or polytene salivary gland cells, but in cellular blastoderm, postblastoderm embryo, and amitotic adult head nuclei, it occurs with a frequency of roughly 4 x 10(5) molecules per nucleus. We used pulse-labeling to study the relationship between H1 synthesis and modification in cultured cells. These results reveal that the H1-associated phosphate is stable and suggest that Drosophila H1 is synthesized, translocated to the nucleus, associated with chromatin, and then phosphorylated. Partial tryptic digestion of Drosophila H1 revealed that the phosphorylation site is located within the globular, central domain of the protein. Thus, the developmentally regulated phosphorylation of Drosophila H1 presents two contrasts with previously studied H1 phosphorylation. It is not correlated with DNA replication, and it is located in the central domain of the protein.     

Characterization and gene cloning of neurotactin, a Drosophila transmembrane protein related to cholinesterases.

Monoclonal antibodies have served to characterize neurotactin, a novel Drosophila protein for which a role in cell adhesion is postulated. Neurotactin is a transmembrane protein, as indicated by epitope mapping and amino acid sequence. Similarly to other cell adhesion molecules, neurotactin accumulates in parts of the membrane where neurotactin-expressing cells contact each other. The protein is only detected during cell proliferation and differentiation, and it is found mainly in neural tissue and also in mesoderm and imaginal discs. Neurotactin has a large cytoplasmic domain rich in charged residues and an extracellular domain similar to cholinesterase that lacks the active site serine required for esterase activity. The extracellular domain also contains three copies of the tripeptide leucine-arginine-glutamate, a motif that forms the primary sequence of the adhesive site of vertebrate s-laminin.     

DRBP76, a double-stranded RNA-binding nuclear protein, is phosphorylated by the interferon-induced protein kinase, PKR.

The interferon-induced double-stranded RNA-activated protein kinase PKR is the prototype of a class of double-stranded (dsRNA)-binding proteins (DRBPs) which share a dsRNA-binding motif conserved from Drosophila to humans. Here we report the purification of DRBP76, a new human member of this class of proteins. Sequence from the amino terminus of DRBP76 matched that of the M phase-specific protein, MPP4. DRBP76 was also cloned by the yeast two-hybrid screening of a cDNA library using a mutant PKR as bait. Analysis of the cDNA sequence revealed that it is the full-length version of MPP4, has a bipartite nuclear localization signal, two motifs that can mediate interactions with both dsRNA and PKR, five epitopes for potential M phase-specific phosphorylation, two potential sites for phosphorylation by cyclin-dependent kinases, a RG2 motif present in many RNA-binding proteins and predicts a protein of 76 kDa. DsRNA and PKR interactions of DRBP76 were confirmed by analysis of in vitro translated and purified native proteins. Cellular expression of an epitope-tagged DRBP76 demonstrated its nuclear localization, and its co-immunoprecipitation with PKR demonstrated that the two proteins interact in vivo. Finally, purified DRBP76 was shown to be a substrate of PKR in vitro, indicating that this protein's cellular activities may be regulated by PKR-mediated phosphorylation.     

Plasma membrane localization of the Toll protein in the syncytial Drosophila embryo: importance of transmembrane signaling for dorsal-ventral pattern formation

Formation of the Drosophila embryo's dorsal-ventral pattern requires the maternal product of the Toll gene. DNA sequence and genetic analyses together suggested that the Toll gene product is a transmembrane protein which communicates information from an extracytoplasmic compartment to the cytoplasm. Using antibodies as probes, we show that the Toll protein is a 135 x 10(3) Mr glycoprotein which is tightly associated with embryonic membranes. During the syncytial stage when dorsal-ventral polarity is established, the maternal Toll protein is associated with the plasma membrane around the entire embryo. During later embryonic stages, the Toll protein is expressed zygotically on many cell surfaces, possibly to promote cell adhesion. The plasma membrane localization of the Toll protein in the syncytial embryo suggests that transmembrane signaling from the extracellular perivitelline space to the cytoplasm is required for establishment of the embryonic dorsal-ventral pattern.     

The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Reconstituted mitochondrial transhydrogenase is a transmembrane protein

Bovine heart mitochondrial transhydrogenase, a redox-linked proton pump, can be functionally and asymmetrically inserted into liposomes by a cholate-dialysis procedure such that the active site faces the external medium. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), a membrane-impermeant photoprobe, when encapsulated in the vesicles, covalently modified the enzyme and inhibited transhydrogenation between NADPH and the 3-acetylpyridine analog of NAD+ (AcPyAD+) in a light-dependent manner. External AcPyAD+ increased the rate of inactivation several fold, whereas NADPH, NADP+, and NADH were without effect. Labeling of the enzyme by intravesicular [35S]NAP-taurine was enhanced by AcPyAD+ and NADP+, decreased by NADH, and not significantly affected by NADPH. These results indicate that transhydrogenase spans the membrane and that substrate binding alters the conformation of that domain of the enzyme protruding from the inner surface of the membrane.     

Vitronectin is phosphorylated by a cAMP-dependent protein kinase released by activation of human platelets with thrombin

Activation of freshly isolated human platelets with a physiological stimulant (thrombin) causes them to release a cAMP-dependent protein kinase which specifically phosphorylates one plasma protein (Mr 75000). This protein is immunochemically and biochemically identified as vitronectin (also know as S protein), which was previously implicated in blood clotting, complement function and cell adhesion.