Two splice variants of Golgi-microtubule-associated protein of 210 kDa (GMAP-210) differ in their binding to the cis-Golgi network.

GMAP-210 (Golgi-microtubule-associated protein of 210 kDa) is a peripheral Golgi protein that interacts with the minus end of microtubules through its C-terminus and with cis-Golgi network membranes through its N-terminus; it participates in the maintenance of the structural integrity of the Golgi apparatus [Infante, Ramos-Morales, Fedriani, Bornens and Rios (1999) J. Cell Biol. 145, 83--98]. We report here the cloning of a new isoform of GMAP-210 that lacks amino acid residues 105--196. On the basis of the analysis of the gmap-210 genomic sequence, we propose that the small isoform, GMAP-200, arises from alternative splicing of exon 4 of the primary transcript. Overexpression of GMAP-200 induces perturbations in both the Golgi apparatus and the microtubule network that are similar to those previously reported for GMAP-210 overexpression. We show that both isoforms are able to oligomerize under overexpression conditions. Analysis in vitro and in vivo, with the green fluorescent protein as a marker, reveals that the binding of the N-terminal domain of GMAP-200 to the cis-Golgi network membranes is lower than that of the N-terminal domain of GMAP-210. Implications for the regulation of interaction between the cis-Golgi network and microtubules are discussed.

Human securin, hPTTG, is associated with Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase.

We have previously isolated the hpttg proto-oncogene, which is expressed in normal tissues containing proliferating cells and in several kinds of tumors. In fact, expression of hPTTG correlates with cell proliferation in a cell cycle-dependent manner. Recently it was reported that PTTG is a vertebrate analog of the yeast securins Pds1 and Cut2, which are involved in sister chromatid separation. Here we show that hPTTG binds to Ku, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). hPTTG and Ku associate both in vitro and in vivo and the DNA-PK catalytic subunit phosphorylates hPTTG in vitro. Furthermore, DNA double-strand breaks prevent hPTTG-Ku association and disrupt the hPTTG-Ku complexes, indicating that genome damaging events, which result in the induction of pathways that activate DNA repair mechanisms and halt cell cycle progression, might inhibit hPTTG-Ku interaction in vivo. We propose that hPTTG might connect DNA damage-response pathways with sister chromatid separation, delaying the onset of mitosis while DNA repair occurs.

Cell cycle regulated expression and phosphorylation of hpttg proto-oncogene product.

We recently isolated a cDNA for hpttg, the human homolog of rat pituitary tumor transforming gene. Now we have analysed the expression of hpttg as a function of cell proliferation. hPTTG protein level is up-regulated in rapidly proliferating cells, is down-regulated in response to serum starvation or cell confluence, and is regulated in a cell cycle-dependent manner, peaking in mitosis. In addition, we show that hPTTG is phosphorylated during mitosis. Immunodepletion and in vitro phosphorylation experiments, together with the use of a specific inhibitor, indicate that Cdc2 is the kinase that phosphorylates hPTTG. These results suggest that hpttg is induced by, and may have a role in, regulatory pathways involved in the control of cell proliferation.

hpttg is over-expressed in pituitary adenomas and other primary epithelial neoplasias.

The role of oncogenes in pituitary tumorigenesis remains elusive since few genetic changes have been identified so far in pituitary tumors. Pituitary tumor-transforming gene (pttg) has been recently cloned from rat GH4 pituitary tumor cells. We have previously isolated and characterized hpttg from human thymus. In the present study, we analyse the expression of hpttg mRNA in a series of human pituitary adenomas. We show that hpttg is highly expressed in the majority of pituitary adenomas while only very low levels of mRNA can be detected in normal pituitary gland by Northern blot analysis. hPTTG protein was immunolocalized mainly in the cytoplasm of adenoma cells. Other common extra-cranial malignant tumors were also analysed by immunohistochemistry. Interestingly, strong hPTTG immunoreactivity was detected in most adenocarcinomas of mammary and pulmonary origins.

GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein.

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1, 979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.

hpttg, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG.

We have isolated a human cDNA clone encoding a novel protein of 22 kDa that is a human counterpart of the rat oncoprotein PTTG. We show that the corresponding gene (hpttg) is overexpressed in Jurkat cells (a human T lymphoma cell line) and in samples from patients with different kinds of hematopoietic malignancies. Analysis of the sequence showed that hPTTG has an amino-terminal basic domain and a carboxyl-terminal acidic domain, and that it is a proline-rich protein with several putative SH3-binding sites. Subcellular fractionation studies show that, although hPTTG is mainly a cytosolic protein, it is partially localized in the nucleus. In addition we demonstrate that the acidic carboxyl-terminal region of hPTTG acts as a transactivation domain when fused to a heterologous DNA binding domain, both in yeast and in mammalian cells.

Arg777 plays a major role in the conformation of the colony-stimulating factor-1 receptor intracellular kinase domain.

A point mutation substituting Arg777 by Gln was obtained in a highly conserved region of the human colony-stimulating factor-1 receptor (CSF-1R) sequence. Constitutive expression of wild-type receptors in CHO cells confers susceptibility to CSF-1 for proliferation whereas the mutated receptors exhibited a 90% reduced efficiency in proliferation. We sought to determine the alterations intervening in the CSF-1 signal transduction of the Arg777Gln mutated receptor. We found that ligand binding and ligand-induced CSF-1R internalization were unaffected. CSF-1-induced receptor dimerization and autophosphorylation were impaired to the same extent as mitogen-activated protein kinase activation (90%). However, only phosphatidylinositol 3-kinase activation and ligand-induced receptor ubiquitination were abrogated by the mutation. These features probably reflect the inability of the mutated CSF-1R kinase domain to fold properly and hence to autophosphorylate and/or to associate correctly with transduction proteins. These data may indicate a role for the conserved regions of the RTK kinase domains in the stabilization of the intracellular domain conformation.

Grb2 and its apoptotic isoform Grb3-3 associate with heterogeneous nuclear ribonucleoprotein C, and these interactions are modulated by poly(U) RNA.

Grb2 is an adaptor molecule comprising one Src homology (SH) 2 and two SH3 domains. This protein has a natural isoform named Grb3-3 with a deletion within the SH2 domain. Numerous evidence points to a functional connection between SH2- and SH3-containing proteins and molecules implicated in RNA biogenesis. In this context, we have examined the binding of Grb2 and Grb3-3 to heterogeneous nuclear ribonucleoprotein (hnRNP) C. By the use of an in vivo genetic approach and through in vitro experiments, we furnish evidence that both Grb2 and Grb3-3 interact with hnRNP C proteins. Subcellular fractionation studies clearly show that Grb2 is partially localized in the nucleus. In addition, coimmunoprecipitation experiments demonstrate that Grb2.hnRNP C complexes exist in intact hematopoietic cells. The carboxyl-terminal SH3 domains of Grb2 and Grb3-3 are primarily responsible for the association with hnRNP C. However, although the proline-rich motif of hnRNP C is involved in the interaction with Grb2, it is not in the binding to Grb3-3. Furthermore, poly(U) RNA inhibits the association of Grb2 with hnRNP C, whereas it enhances the interaction between Grb3-3 and hnRNP C. These findings suggest that the Grb2/Grb3-3-hnRNP C interactions might fulfill different biological functions.

NA14 is a novel nuclear autoantigen with a coiled-coil domain.

The serum from a patient with Sjögren's syndrome (RM serum) was used to screen a human testis cDNA expression library. A cDNA of 865 base pairs containing the entire coding sequence for a novel protein was isolated. The 14-kDa predicted protein contains an acidic domain (amino acids 6-80) with a high frequency of heptad repeats characteristic of alpha-helices that form dimeric coiled-coil structures and an alkaline carboxyl-terminal domain (amino acids 81-119). It seems to be widely expressed, but its expression level varies depending on tissues. A protein of apparent molecular mass of 14 kDa was immunoprecipitated from cell lysates by the autoimmune serum, and it was recognized by rabbit antibodies raised to a recombinant bacterial fusion protein generated from the cDNA clone. Conventional and confocal immunofluorescence microscopy on HeLa and 3T3 cells transiently transfected with a tagged form of the protein showed numerous punctate structures scattered throughout the nucleus. This novel protein has been termed NA14 for Nuclear Autoantigen of 14 kDa.

Adenosine deaminase is a specific partner for the Grb2 isoform Grb3-3.

Grb3-3 is an isoform of Grb2, thought to arise by alternative splicing, that lacks a functional SH2 domain but retains functional SH3 domains, which allow interaction with other proteins through binding to prolinerich sequences. Several evidences suggest that besides common partners for Grb2 and Grb3-3, specific targets could exist. In order to find specific partners for Grb3-3, we have screened a human cDNA library by the yeast two-hybrid system with Grb3-3 as a bait. We have identified adenosine deaminase, an enzyme involved in purine metabolism whose deficiency is associated with severe combined immunodeficiency, as a Grb3-3 binding protein that is not able to bind to Grb2. This interaction has been confirmed in vitro with GST fusion proteins and in vivo by coimmunoprecipitation experiments in NIH3T3 cells stably transfected with Grb3-3. The functional significance of this finding is discussed.

A role for JNK/SAPK in proliferation, but not apoptosis, of IL-3-dependent cells.

Activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) has been implicated in the induction of apoptosis in a variety of systems [1] [2] [3] [4] [5] [6] [7] [8]. BAF3 cells are pre-B cells that undergo apoptosis following IL-3 withdrawal or ceramide treatment [9] [10]. JNK/SAPK in BAF3 cells is stimulated by ceramide and also during cell proliferation in response to IL-3 [11], but its role in the apoptotic response is not clear. We have devised a method of selectively inhibiting JNK/SAPK activity using a dual-specificity threonine/tyrosine phosphatase, M3/6. Expression of this phosphatase in BAF3 cells prevented ceramide stimulation of JNK/SAPK activity but did not affect apoptosis following IL-3 withdrawal or ceramide treatment. IL-3-stimulated proliferation of BAF3 cells expressing the phosphatase was, however, inhibited. Hence JNK/SAPK activation is likely to be involved in the proliferative response of these cells but is not required for apoptosis. Selective ablation by dual-specificity phosphatases should be a general method for determining the functions of specific mitogen-activated kinase pathways.

Association of the protein tyrosine phosphatase PTP1C with the protein tyrosine kinase c-Src in human platelets.

Protein tyrosine phosphatase 1C (PTP1C), highly expressed in hematopoietic cells, is a soluble protein tyrosine phosphatase containing two Src homology 2 (SH2) domains at the N-terminus and two putative sites of tyrosine phosphorylation at the C-terminus. This paper reports that PTP1C and c-Src could be coimmunoprecipitated during thrombin-induced platelet activation. Moreover, association between the two signalling proteins occurred only after PTP1C had been tyrosine phosphorylated. In in vitro experiments, PTP1C bound to the SH2 domain of c-Src, suggesting that association between tyrosine phosphorylated PTP1C and c-Src was mediated by the SH2 domain of c-Src. Finally, in resting platelets, PTP1C was mainly found in the Nonidet P-40 soluble fraction whereas following thrombin-induced activation, around 17% of PTP1C was associated with the insoluble fraction.

The proline-rich region of Vav binds to Grb2 and Grb3-3.

Vav has structural features found in signaling proteins and is expressed only in hematopoietic cells. The recent development of mice Vav -/- has confirmed a major role of Vav in early blood cell development. We previously showed that Vav constitutively interacts with glutathione-S-transferase-Grb2. Coimmunoprecipitation experiments supported the idea of a complex formed by Vav-Grb2 in vivo. This complex is of potential interest in signaling of hematopoietic cells. In this work we localize the domains of Vav and Grb2 involved in this interaction. By the use of an in vivo genetic approach (the double hybrid system) and through in vitro experiments (glutathione-S-transferase fusion proteins) we furnish evidence that the interaction between Vav and Grb2 involves the C-SH3 domain of Grb2 and the proline-rich region located in the N-SH3 of Vav. Furthermore this was confirmed by the use of both Vav and Sos derived proline-rich peptides which blocked the binding. In addition we show that Vav also interacts with Grb3-3, a naturally occurring Grb2 isoform wich lacks functional SH2 domain.

In vitro binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase.

Although the human immunodeficiency virus type 1 (HIV-1) nef gene still has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1LAI nef gene product was expressed in the bacterial vector pGEX-2T as a glutathione S-transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites in vitro: one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef in vitro. The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis.

Naturally occurring anti-idiotypic antibodies to anti-phosphotyrosine in systemic lupus erythematosus interact with SRC-homology 2 domains.

We have recently identified in SLE sera naturally occurring anti-idiotypic antibodies against anti-phosphotyrosine antibodies. Analysis of immunochemical properties of these anti-idiotypic antibodies suggest that they are of beta/gamma type mimicking the antigen. The interaction between these anti-idiotypes and SH2 domains of various fusion proteins was analysed by immunoprecipitation and immunoblotting. Our data demonstrate that these anti-idiotypic antibodies specifically bind SH2 domains, with the highest affinity for SH2 domain of lck protein tyrosine kinase. The significance of this interaction is discussed.

Endothelin induces tyrosine phosphorylation and GRB2 association of Shc in astrocytes.

While the mitogen-activated protein kinase (MAPK) pathway coupled to receptor tyrosine kinases has been largely clarified, little is known about MAPK activation mediated by heterotrimeric G protein-coupled receptors. In a previous study, it has been shown that endothelin-1 (ET-1) signaling through heterotrimeric G protein-coupled receptors stimulates MAPK activity in primary cultures of astrocytes (Cazaubon, S., Parker, P. J., Strosberg, A.D., and Couraud, P.O. (1993) Biochem. J. 293, 381-386). To clarify the molecular mechanism responsible for this response, involvement of the adapter proteins, Shc and Grb2, has now been investigated. It is shown here that in these cells, ET-1 stimulates tyrosine phosphorylation of Shc, resulting in its stable association with Grb2 but not with Grb3-3, a Grb2 isoform with partially deleted SH2 domain. These results demonstrate that tyrosine-phosphorylated Shc specifically interacts with the SH2 domain of Grb2. This response was rapid and transient, showing a maximum at 10 min and declining at 60 min. Interestingly, direct activation of G proteins by fluoroaluminate mimics the ET-1 effect. In addition, a shift to a higher apparent molecular mass of Raf-1 kinase, likely reflecting its hyperphosphorylation, was also detected in ET-1-treated cells. These data strongly suggest that ET-1-induced MAPK activation is a G protein-coupled pathway that involves Shc, Grb2, and probably Raf-1. In conclusion, the Shc-Grb2 complex may be involved in the activation of the MAPK pathway, not only by several receptor tyrosine kinases but also by heterotrimeric G protein-coupled receptors, such as ET-1 receptors.

P56lck A lymphocyte specific protein tyrosine kinase: activation, regulation and signal transduction.

p56lck, a src family protein tyrosine kinase interacts with several T cell receptors, like: CD4, CD8, CD2 and the beta-chain of the IL2, thereby receptors devoid of kinase activity may transduce signals via tyr phosphorylation. Tyr 192 and ser 194, located in the SH2 domain of p56lck is phosphorylated upon CD3 triggering, which can change interactions of tyr-P proteins with this SH2 domain. Upon activation through the CD2 or the CD45 receptors the kinase activity of p56lck is temporarily increased. By immunofluorescent and confocal microscopy we observed that a significant proportion of p56lck and CD2 receptors are localized in endosomal vesicles after stimulation. By Western blot we showed a parallel recruitment of the PTK p70-ZAP in this vesicles. The role of p56lck away from the plasma membrane localized in vesicles is under study.

Vav binds to several SH2/SH3 containing proteins in activated lymphocytes.

In T lymphocytes, several proteins are rapidly phosphorylated on tyrosine after stimulation. In this study we examine the ability of tyrosine phosphorylated proteins from Jurkat T cells stimulated by CD2 or T cell receptor-CD3 to interact with the src homology 2 or src homology 3 domains from eight different proteins involved in signal transduction in lymphocytes: Vav, Shc, Nck, phosphatidylinositol-3-kinase, phospholipase C-gamma 1, Ras-GTPase activating protein, c-Crk and Grb2. Our data show that different SH2 domains have distinct patterns of binding to phosphotyrosine containing proteins. We show that Vav, a protein expressed only in hematopoietic cells that may have guanine nucleotide releasing factor activity, is able to interact with certain SH2-containing proteins depending on its tyrosine phosphorylation and with Grb2 in a manner independent of phosphorylation on tyrosine. Coimmunoprecipitation experiments support the idea of a trimolecular complex Shc-Grb2-Vav in vivo. These data suggest a central role played by Vav and provide insight in the complexity and specificity of protein-protein interactions in the signaling events in lymphocytes.

P56lck: a transducing protein that binds to SH2 containing proteins and to phosphotyrosine containing proteins.

In T lymphocytes, several proteins are rapidly phosphorylated on tyrosine after stimulation. In this study we examine the ability of tyrosine phosphorylated proteins from Jurkat T cells stimulated by CD2 or T cell receptor (TcR)-CD3 to interact with the src homology 2 (SH2) domains from p56lck (Lck). Our data show that the patterns are different depending on the stimulation. The specificity of the interactions was assessed by blocking experiments with high affinity phosphotyrosine [Y(P)] peptides. Phosphorylation experiments suggest that one or several kinases are able to interact with the SH2 from Lck. On the other hand, full length Lck overexpressed in Sf9 cells, which is tyrosine-phosphorylated at least on two sites, can interact in vitro with the SH2 from Lck, phospholipase C (PLC)-gamma 1, p85 (the regulatory subunit of phosphatidyl-inositol-3 kinase (PI3K)) and Nck and with the full length Grb2. These data give additional support to the idea that Lck is an important signal transducing molecule in lymphocytes.