Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1

SHP-1 is an SH2-containing cytoplasmic tyrosine phosphatase that is widely distributed in cells of the hematopoietic system. SHP-1 plays an important role in the signal transduction of many cytokine receptors, including the receptor for erythropoietin, by associating via its SH2 domains to the receptors and dephosphorylating key substrates. Recent studies have suggested that SHP-1 regulates the function of Jak family tyrosine kinases, as shown by its constitutive association with the Tyk2 kinase and the hyperphosphorylation of Jak kinases in the motheaten cells that lack functional SHP-1. We have examined the interactions of SHP-1 with two tyrosine kinases activated during engagement of the erythropoietin receptor, the Janus family kinase Jak-2 and the c-fps/fes kinase. Immunoblotting studies with extracts from mouse hematopoietic cells demonstrated that Jak2, but not c-fes, was present in anti-SHP-1 immunoprecipitates, suggesting that SHP-1 selectively associates with Jak2 in vivo. Consistent with this, when SHP-1 was coexpressed with these kinases in Cos-7 cells, it associated with and dephosphorylated Jak2 but not c-fes. Transient cotransfection of truncated forms of SHP-1 with Jak2 demonstrated that the SHP-1-Jak2 interaction is direct and is mediated by a novel binding activity present in the N terminus of SHP-1, independently of SH2 domain-phosphotyrosine interaction. Such SHP-1-Jak2 interaction resulted in induction of the enzymatic activity of the phosphatase in in vitro protein tyrosine phosphatase assays. Interestingly, association of the SH2n domain of SHP-1 with the tyrosine phosphorylated erythropoietin receptor modestly potentiated but was not essential for SHP-1-mediated dephosphorylation of Jak2 and had no effect on c-fes phosphorylation. These data indicate that the main mechanism for regulation of Jak2 phosphorylation by SHP-1 involves a direct, SH2-independent interaction with Jak2 and suggest the existence of similar mechanisms for other members of the Jak family of kinases. They also suggest that such interactions may provide one of the mechanisms that control SHP-1 substrate specificity.     

Suppression of GnRH gene expression in GT1-1 hypothalamic neuronal cells: action of protein kinase C

We attempted to elucidate molecular mechanisms of gonadotropin-releasing hormone (GnRH) gene regulation by the protein kinase C (PKC) pathway in GT1-1 cells. Activation of PKC with 12-tetra-decanoylphorbol-13-acetate (TPA) or inhibition with staurosporine or calphostin C down-regulated GnRH mRNA levels. A serial deletion mutant analysis revealed that this suppression was mediated by the proximal region (-187/-69) of the mouse GnRH promoter. TPA transiently induced c-fos mRNA, whereas staurosporine or calphostin C failed to do so. However, PKC inhibitors blocked the TPA-evoked c-fos induction. Over-expression of PKC alpha down-regulated GnRH promoter activity, indicating that PKC activation was sufficient to inhibit GnRH gene expression. These results suggest that both activation and inhibition of PKC decrease the GnRH gene expression in the GT1-1 cells probably through different signal cascade mechanisms.     

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.     

Selective neuronal expression of green fluorescent protein with cytomegalovirus promoter reveals entire neuronal arbor in transgenic mice

In simple nervous systems, identified groups of neurons can be studied in depth. To allow the same advantage in the mammalian brain, we have generated green fluorescent protein (GFP) transgenic mice in which only a few types of neurons are strongly labeled with a fluorescent molecule, which the neurons synthesize internally, allowing the cells, their dendrites, filopodia, and axons to be identified in both living and fixed CNS, in slices and culture. The same neurons, with GFP expression controlled by part of the major immediate early promoter of human cytomegalovirus (CMV), show GFP beginning early in development, from one generation to the next, allowing cellular and physiological studies of axonal and dendritic growth, fate mapping, anatomical connections, and synapse formation in identified neurons. The human CMV promoter sequence we used was different from that used in previous work with other reporter genes and gave a dramatically different pattern of expression. Two transgenic lines with the same CMV promoter show similar anatomical patterns of expression in the present study. Strong GFP labeling was found in a subpopulation of mossy fibers that innervated parasagittal bands in the cerebellar cortex and olfactory axons that projected into the olfactory bulb, subsets of motoneurons and dorsal root ganglion cells, granule but not mitral cells of the olfactory bulb, and a group of neurons in the hypothalamic suprachiasmatic nucleus. A novel type of neuron was strongly labeled in the olfactory bulb external plexiform layer. In normal brains, CMV does not constitute a threat, but in the developing brain, CMV can cause debilitating neurodegeneration and death; studies using the CMV promoter aid in understanding the affinity of CMV that has been suggested for specific brain regions.     

Neuronal pentraxin receptor, a novel putative integral membrane pentraxin that interacts with neuronal pentraxin 1 and 2 and taipoxin-associated calcium-binding protein 49

We have identified the first putative integral membrane pentraxin and named it neuronal pentraxin receptor (NPR). NPR is enriched by affinity chromatography on columns of a snake venom toxin, taipoxin, and columns of the taipoxin-binding proteins neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and taipoxin-associated calcium-binding protein 49 (TCBP49). The predominant form of NPR contains an putative NH2-terminal transmembrane domain and all forms of NPR are glycosylated. NPR has 49 and 48% amino acid identity to NP1 and NP2, respectively, and NPR message is expressed in neuronal regions that express NP1 and NP2. We suggest that NPR, NP1, NP2, and TCBP49 are involved in a pathway responsible for the transport of taipoxin into synapses and that this may represent a novel neuronal uptake pathway involved in the clearance of synaptic debris.     

Kidney-specific expression of a novel mouse organic cation transporter-like protein

OBJECTIVE: To assess the anterior mediastinal mass in recurrent testicular cancer, with relation to thymic hyperplasia after treatment. METHODS: The anterior mediastinal regions were fully evaluated by chest computed tomography (CT) at the initial staging and after treatment in 24 of 44 patients with testicular cancer. RESULTS: One patient with stage IIB tumor had thymic hyperplasia before treatment, and one with stage III had benign thymic hyperplasia after chemotherapy with salvage surgery. Three of 4 patients who had recurrence had an anterior mediastinal mass. One had benign thymic hyperplasia confirmed by histology and 2 had metastatic tumor confirmed by histology and clinical course, in which the mass became so enlarged that it obstructed major vessels. CONCLUSION: Although the relationship of the CT finding to the response to treatment in the anterior mediastinal mass and other metastatic lesions provide some clues helpful in differentiating benign from malignant masses, surgical exploration is recommended for the patient with an indication for salvage surgery.     

C2H2-171: a novel human cDNA representing a developmentally regulated POZ domain/zinc finger protein preferentially expressed in brain

We describe a novel human zinc finger cNDA. C2H2-171. This cDNA represents an mRNA which encodes a protein of 484 amino acids and a calculated molecular weight of 54 kD. Four zinc finger-like domains are found in the C-terminal end of the protein. At the N-terminus, C2H2-171 contains a POZ/tramtrack-like domain similar to that found in the tumor associated zinc finger proteins LAZ-3/BCL-6 and PLZ-F, as well as in non-zinc finger proteins. C2H2-171 RNA is preferentially expressed in the brain, and increases during the course of murine development, with maximal expression in the adult. C2H2-171 RNA is differentially expressed in brain regions, with the highest level of expression in the cerebellum. C2H2-171 RNA was expressed at high levels in primary cerebellar granule cell neurons compared to astrocytes. The gene encoding C2H2-171 is highly conserved in vertebrates, and maps to the terminus of human chromosome 1 (1q44-ter). This chromosomal location is associated with a number of cytogenetic aberrations including those involving brain developmental anomalies and tumorigenesis. These data suggest that C2H2-171 may play an important role in vertebrate brain development and function.     

Identification of a novel Rac3-interacting protein C1D

The actin cytoskeleton is an important contributor to the integrity of cellular shape and responses in neurons. However, the molecular mechanisms associated with functional interactions between the actin cytoskeleton and neuronal ion channels are largely unknown. Whole-cell and single channel recording techniques were thus applied to identified retinal bipolar neurons of the tiger salamander (Ambystoma tigrinum) to assess the role of acute changes in actin-based cytoskeleton dynamics in the regulation of voltage-gated ion channels. Disruption of endogenous actin filaments after brief treatment (20-30 min) with cytochalasin D (CD) activated voltage-gated K+ currents in bipolar cells, which were largely prevented by intracellular perfusion with the actin filament-stabilizer agent, phalloidin. Either CD treatment under cell-attached conditions or direct addition of actin to excised, inside-out patches of bipolar cells activated and/or increased single K+ channels. Thus, acute changes in actin-based cytoskeleton dynamics regulate voltage-gated ion channel activity in bipolar cells.     

Microtubule-associated protein-2 in the rat testis: a novel site of expression

The testis is one of the most abundant sources of microtubule networks. These networks include mitotic and meiotic spindles, the spermatid manchette and axoneme, and the Sertoli cell cytoskeleton. Microtubules are composed of alpha- and beta-tubulin subunits that are polymerized and stabilized by a variety of microtubule-associated proteins (MAPs). One of these, MAP2, has been extensively characterized as a brain-specific protein with the capacity to bind tubulin, cAMP-dependent kinase, and calmodulin. MAP2 mRNA is processed into at least two variants encoding proteins designated MAP2a, MAP2b, and MAP2c. Of the 5.7 kb of coding sequence in the 9-kb mRNA that encodes MAP2a and MAP2b, a deletion of approximately 4 kb produces mRNA encoding MAP2c, which consists of only the N- and C- terminal regions of MAP2b. To determine whether MAP2 was present in the rat testis, microtubule preparations were isolated from adult rat testis and brain by means of taxol-mediated polymerization and analyzed by gel filtration, ELISA, and Western blotting using polyclonal and monoclonal antibodies reactive with MAP2. A 74-kDa protein corresponding to MAP2c was detected in the testis. These results were confirmed by Northern blot analysis of total RNA from adult rat brain and testis with cDNA probes that distinguish between the known MAP2 splice variants. The predominant mRNAs in testis of 6 kb and 2.5-3.5 kb corresponded to MAP2c. A single 6-kb mRNA with the potential to encode MAP2c was detected in enriched preparations of immature Sertoli cells and adult Leydig cells. Round spermatids contained at least two MAP2 mRNAs between approximately 2.5 and 3.5 kb in size that displayed a stage-specific pattern of expression. Immunohistochemistry showed a MAP2-like protein in both somatic and germ cells, with a particularly distinct localization within the cytoplasm of primary and secondary spermatocytes at stage XIV of the seminiferous cycle during meiotic metaphase. In addition to cytoplasmic staining, a novel localization of this protein was observed in the nucleus of many testicular cells.     

Synaptojanin 2, a novel synaptojanin isoform with a distinct targeting domain and expression pattern

Synaptojanin (synaptojanin 1) is a recently identified inositol 5'-phosphatase, which is highly enriched in nerve terminals and is implicated in synaptic vesicle recycling. It is composed of three domains: an amino-terminal SacI homology region, a central inositol 5'-phosphatase homology region, and a carboxyl-terminal proline-rich region. We have now identified and characterized a novel form of synaptojanin, synaptojanin 2, which has a broader tissue distribution. Synaptojanin 2 cDNA from rat brain library encodes a protein of 1,248 amino acids with a predicted Mr of 138,268. The two synaptojanin isoforms share 57.2 and 53.8% amino acid identity in their SacI and phosphatase domains, respectively. In marked contrast, their carboxyl-terminal proline-rich regions bear little homology. Expression of synaptojanin 2 in COS7 cells produced a 140-kDa protein with inositol 5'-phosphatase actvity. Protein binding assays demonstrated that among the major src homology 3-proteins known to bind to the proline-rich region of synaptojanin 1, Grb2, amphiphysin, and members of SH3p4/8/13 protein family, only Grb2 bound to that of synaptojanin 2. Furthermore, subcellular fractionation studies in transfected Chinese hamster ovary cells revealed that synaptojanin 2 was predominantly associated with the particulate fraction while synaptojanin 1 was mainly localized in the soluble fraction. This observation suggests that the proline-rich regions of synaptojanins 1 and 2 are implicated in different protein-protein interactions and direct the two isoforms to different subcellular compartments. Our results demonstrate the presence of a family of synaptojanin-type inositol 5'-phosphatases with different tissue and subcellular distributions, which may be involved in distinct membrane trafficking and signal transduction pathways in mammalian cells.     

Synergistic regulation of a neuronal chloride current by intracellular calcium and muscarinic receptor activation: a role for protein kinase C

Using perforated patch recordings in combination with intracellular Ca2+ ([Ca2+]i) fluorescence measurements, we have identified a delayed Ca(2+)-dependent Cl- current in a mammalian sympathetic ganglion cell. This Cl- current is induced by the synergistic action of Ca2+ and diacylglycerol (DAG) and is blocked by inhibitors of protein kinase C. As a result, the current can be induced by acetylcholine through the conjoint activation of nicotinic receptors (to produce a rise in [Ca2+]i) and muscarinic receptors (to generate DAG). This demonstrates an unusual form of synergism between the two effects of a single transmitter mediated via separate receptors operating within a time scale that could be of physiological significance.     

Localization of Niemann-Pick C1 protein in astrocytes: implications for neuronal degeneration in Niemann- Pick type C disease

Niemann-Pick type C disease (NP-C) is an inherited neurovisceral lipid storage disorder characterized by progressive neurodegeneration. Most cases of NP-C result from inactivating mutations of NPC1, a recently identified member of a family of genes encoding membrane-bound proteins containing putative sterol sensing domains. By using a specific antipeptide antibody to human NPC1, we have here investigated the cellular and subcellular localization and regulation of NPC1. By light and electron microscopic immunocytochemistry of monkey brain, NPC1 was expressed predominantly in perisynaptic astrocytic glial processes. At a subcellular level, NPC1 localized to vesicles with the morphological characteristics of lysosomes and to sites near the plasma membrane. Analysis of the temporal and spatial pattern of neurodegeneration in the NP-C mouse, a spontaneous mutant model of human NP-C, by amino-cupric-silver staining, showed that the terminal fields of axons and dendrites are the earliest sites of degeneration that occur well before the appearance of a neurological phenotype. Western blots of cultured human fibroblasts and monkey brain homogenates revealed NPC1 as a 165-kDa protein. NPC1 levels in cultured fibroblasts were unchanged by incubation with low density lipoproteins or oxysterols but were increased 2- to 3-fold by the drugs progesterone and U-18666A, which block cholesterol transport out of lysosomes, and by the lysosomotropic agent NH4Cl. These studies show that NPC1 in brain is predominantly a glial protein present in astrocytic processes closely associated with nerve terminals, the earliest site of degeneration in NP-C. Given the vesicular localization of NPC1 and its proposed role in mediating retroendocytic trafficking of cholesterol and other lysosomal cargo, these results suggest that disruption of NPC1-mediated vesicular trafficking in astrocytes may be linked to neuronal degeneration in NP-C.     

Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.     

Bcl-2 protein as a marker of neuronal immaturity in postnatal primate brain

The distribution of neurons expressing immunoreactivity for the protein Bcl-2 was studied in the brain of squirrel monkeys (Saimiri sciureus) of various ages. Several subsets of small and intensely immunoreactive neurons displaying an immature appearance were disclosed in the amygdala and piriform cortex. The piriform cortex exhibited clusters of various forms in which Bcl-2+ neurons appeared linked to one another by their own neurites. The subventricular zone, which is known to harbor the largest population of rapidly and constitutively proliferating cells in the adult rat brain, was intensely stained, particularly at the basis of the lateral ventricle. A long and dorsoventrally oriented Bcl-2+ fiber fascicle was seen to emerge from the subventricular zone, together with numerous Bcl-2+ cells that formed a densely packed column directed at the olfactory tubercle. In adult and aged monkeys, the small and intensely labeled neurons were progressively replaced by larger and more weakly stained neurons in the amygdala and piriform cortex. In contrast, Bcl-2 immunostaining did not change with age in the subventricular zone and olfactory tubercle, the islands of Calleja of which were markedly enriched with Bcl-2. The dentate gyrus contained only a few layers of intensely labeled granule cells in juvenile monkeys, but the number of these layers increased markedly in adult and aged monkeys. These findings suggest that Bcl-2 can serve as a marker of both proliferating and differentiating neurons and indicate that such immature neurons may be much more widespread than previously thought in postnatal primate brain.