Joan B. Levy

Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases.

The tyrosine phosphatase RPTP gamma is a candidate tumor suppressor gene since it is located on human chromosome 3p14.2-p21 in a region frequently deleted in certain types of renal and lung carcinomas. In order to evaluate its oncogenic potential and to explore its normal in vivo functions, we have isolated cDNAs and deduced the complete sequences of both human and murine RPTP gamma. The murine RPTP gamma gene has been localized to chromosome 14 to a region syntenic to the location of the human gene. Northern (RNA) blot analysis reveals the presence of two major transcripts of 5.5 and 8.5 kb in a variety of murine tissues. In situ hybridization analysis reveals that RPTP gamma mRNA is expressed in specific regions of the brain and that the localization of RPTP gamma changes during brain development. RPTP gamma is composed of a putative extracellular domain, a single transmembrane domain, and a cytoplasmic portion with two tandem catalytic tyrosine phosphatase domains. The extracellular domain contains a stretch of 266 amino acids with striking homology to the zinc-containing enzyme carbonic anhydrase (CAH), indicating that RPTP gamma and RPTP beta (HPTP zeta) represent a subfamily of receptor tyrosine phosphatases. We have constructed a model for the CAH-like domain of RPTP gamma based upon the crystal structure of CAH. It appears that 11 of the 19 residues that form the active site of CAH are conserved in RPTP gamma. Yet only one of the three His residues that ligate the zinc atom and are required for catalytic activity is conserved. On the basis of this model we propose that the CAH-like domain of RPTP gamma may have a function other than catalysis of hydration of metabolic CO2.

Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts.

Colony-stimulating factor-1 (CSF-1) stimulates motility and cytoplasmic spreading in mature osteoclasts. Therefore, we examined the cellular events and intracellular signaling pathways that accompany CSF-1-induced spreading in normal osteoclasts. To explore the role c-src plays in these processes, we also studied osteoclasts prepared from animals with targeted disruption of the src gene. In normal osteoclasts, CSF-1 treatment induces rapid cytoplasmic spreading, with redistribution of F-actin from a well-delineated central attachment ring to the periphery of the cell. CSF-1 increases membrane phosphotyrosine staining in osteoclasts and induces the phosphorylation of several cellular proteins in cultured, osteoclast-like cells, including c-fms, c-src, and an 85-kD Grb2-binding protein. Src kinase activity is increased threefold after CSF-1 treatment. In src- cells, no attachment ring is present, and CSF-1 fails to induce spreading or a change in the pattern of F-actin distribution. Although c-fms becomes phosphorylated after CSF-1 treatment, the 85-kD protein is significantly less phosphorylated in src- osteoclast-like cells. These results indicate that c-src is critical for the normal cytoskeletal architecture of the osteoclast, and, in its absence, the spreading response induced by CSF-1 is abrogated, and downstream signaling from c-fms is altered.

The expression of a novel receptor-type tyrosine phosphatase suggests a role in morphogenesis and plasticity of the nervous system

Analysis of the localization of receptor-type protein tyrosine phosphatase-beta (RPTP-beta) by in situ hybridization and immunocytochemistry indicates that it is predominantly expressed in the developing central nervous system (CNS). RPTP-beta is highly expressed in radial glia and other forms of glial cells that play an important role during development. The immunoreactivity localizes to the radial processes of these cells, which act as guides during neuronal migration and axonal elongation. The pattern of RPTP-beta expression changes with the progression of glial cell differentiation. In the adult, high levels of RPTP-beta are seen in regions of the brain where there is continued neurogenesis and neurite outgrowth. The spatial and temporal patterns of RPTP-beta expression suggest that this receptor phosphatase plays a role in morphogenesis and plasticity of the nervous system.

The tyrosine phosphatase SHP-1 is a negative regulator of osteoclastogenesis and osteoclast resorbing activity: increased resorption and osteopenia in mev/mev mutant mice

Naturally occuring inactivating mutations of the Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 (SHP-1) in mice give rise to the motheaten (me) phenotype. me/me mice have multiple hematopoietic abnormalities, suggesting that this phosphatase plays an important role in hematopoiesis. SHP-1 binds to and is activated by several hematopoietic surface receptors, including the colony-stimulating factor type 1 receptor. We have examined the role of SHP-1 in osteoclastogenesis and osteoclast function using mice with the viable motheaten (me(v)/me(v)) mutation, which has markedly decreased SHP-1 activity. Histomorphometric analysis of 6-week-old me(v)/me(v) mice and control littermates showed a marked osteopenia with an increase in bone resorption indices. The number of formed osteoclast-like cells (OCLs) in cocultures of me(v)/me(v) hematopoietic cells with normal osteoblasts was significantly increased. In contrast, the number of OCLs formed in the coculture of normal bone marrow cells with the me(v)/me(v) osteoblasts was not significantly different from controls. The bone-resorbing activity of me(v)me(v) OCLs and authentic osteoclasts was also found to be increased. Finally, Western blotting of proteins from me(v)/me(v) and control OCLs revealed an overall increase in tyrosine phosphorylation in the me(v)/me(v) lysates. These in vivo and in vitro results suggest that SHP-1 is a negative regulator of bone resorption, affecting both the formation and the function of osteoclasts.

Molecular complexes that contain both c-Cbl and c-Src associate with Golgi membranes

Cbl is an adaptor protein that is phosphorylated and recruited to several receptor and non-receptor tyrosine kinases upon their activation. After binding to the activated receptor, Cbl plays a key role as a kinase inhibitor and as an E3 ubiquitin ligase, thereby contributing to receptor down-regulation and internalization. In addition, Cbl translocates to intracellular vesicular compartments following receptor activation. We report here that Cbl also associates with Golgi membranes. Confocal immunofluorescence staining of Cbl in a variety of unstimulated cells, including CHO cells, revealed a prominent perinuclear colocalization of Cbl and a Golgi marker. Both the prominent Cbl staining and the Golgi marker were dispersed by brefeldin A. Subcellular fractionation of CHO cells demonstrated that about 10% of Cbl is stably associated with membranes, and that Golgi-enriched membrane fractions produced by isopycnic density centrifugation and free-flow electrophoresis are also enriched in Cbl, relative to other membrane fractions. The membrane-bound Cbl was hyperphosphorylated and it co-immunoprecipitated with endogenous Src. By immunofluorescence, some Src colocalized with Cbl and Golgi markers, and Src, like Cbl, was present in the Golgi-enriched fraction prepared by sequential density centrifugation and free-flow electrophoresis. Transfection of an activated form of Src, but not wild-type Src, increased the amount of Src that co-immunoprecipitated with Cbl, and increased the intensity of Cbl staining on the Golgi. This result, together with the increased tyrosine phosphorylation of the membrane-associated Cbl, suggests that Golgi-associated Cbl could be part of a molecular complex that contains activated Src. The localization and interaction of Src and Cbl at the Golgi and the regulation of the interaction of Cbl with Golgi membrane suggest that this complex may contribute to the regulation of Golgi function.

Role of c‐Src in cellular events associated with colony‐stimulating factor‐1‐induced spreading in osteoclasts

We and others have observed that in response to treatment with Colony Stimulating Factor‐1 (CSF‐1) neonatal rat osteoclasts demonstrate rapid cytoplasmic spreading. The receptor for CSF‐1, c‐Fms, is expressed in osteoclasts, possesses intrinsic tyrosine‐kinase activity, and signals via rapid phosphorylation of selected proteins. It has been reported previously that c‐Src becomes tyrosine phosphorylated following CSF‐1 treatment of fibroblasts overexpressing c‐Fms. We therefore examined the cellular events associated with CSF‐1‐induced spreading in osteoclasts and what role, if any, c‐Src played in these processes.

Identification of a Novel 135-kDa Grb2-binding Protein in Osteoclasts

The tyrosine kinase receptor for macrophage colony-stimulating factor and the non-receptor tyrosine kinase c-Src play critical roles in osteoclast differentiation and function. Since the ubiquitously expressed adaptor protein Grb2 plays an important role in several tyrosine kinase signal transduction pathways, we used a filter binding assay to identify osteoclast proteins that bind to Grb2. In osteoclasts, there were three major Grb2-binding proteins, two of which, mSos and c-Cbl (p120), have been previously identified as Grb2-binding proteins in many cell types. The third protein, p135, had a restricted pattern of expression and was present at high levels in authentic osteoclasts and osteoclast-like cells formed in an in vitro co-culture system. In addition to binding Grb2 in the filter binding assay, p135 was isolated in complexes with endogenous Grb2 from osteoclast cell extracts. The association of p135 and Grb2 was dependent on an intact Src homology 3 domain and furthermore, was shown to preferentially interact with the N-terminal Src homology 3 domain of Grb2, which is similar to the interaction of mSos and Grb2 in other cell types. p135 was not recognized by antibodies against several known Grb2-binding proteins and thus may be a novel Grb2-binding protein.

Erratum Molecular complexes that contain both c-Cbl and c-Scr associate with Golgi membranes

Summary Eur. J. Cell Biol. 81, 26–35 (2002). Upon request of the auhtors the name of Pierre Jurdic (Laboratoire de Biologie Moleculaire et Cellulaire, UMR 5665 CNRS/ENS, INRA, Ecole Normale Superieure de Lyon, Lyon/France) has to be added to the list of authors between J. B. Levy and W. C. Horne.