p120 GAP requirement in normal and malignant human hematopoiesis.

There is evidence to suggest that the p120 GAP (GAP), originally described as an inhibitor of p21ras, may also serve as a downstream effector of ras-regulated signal transduction. To determine whether GAP expression is required for the growth of human normal and leukemic hematopoietic cells, we used GAP antisense oligodeoxynucleotides to inhibit it and analyzed the effects of this inhibition on the colony-forming ability of nonadherent, T lymphocyte-depleted mononuclear cells and of highly purified progenitors (CD34+ MNC) obtained from the bone marrow and peripheral blood of healthy volunteers or chronic myeloid leukemia (CML, bcr-abl-positive) patients. The acute myelogenous leukemia cell line MO7, the Philadelphia BV173 cell line, and the acute promyelocytic leukemia NB4 and HL-60 cell lines were similarly examined. GAP antisense treatment inhibited colony formation from normal myelo-, erythro-, and megakaryopoietic progenitor cells as well as from CML progenitor cells. Proliferation of MO7 (growth factor-dependent) and BV173 (bcr-abl-dependent) cells, but not that of NB4 and HL-60 (growth factor-independent) cells, was also inhibited, even though a specific downregulation of GAP was observed in each cell line, as analyzed by either or both mRNA and protein expression. Stimulation of MO7 cells with hematopoietic growth factors increased the expression of GAP as well as the levels of active GTP-bound p21ras. Stimulation of GAP expression was inhibited upon GAP antisense treatment. These data indicate that p120 GAP is involved in human normal and leukemic hemopoiesis and strongly suggest that GAP is not only a p21ras inhibitor (signal terminator), but also a positive signal transducer.

Detergent-solubilized RNA polymerase from cells infected with foot-and-mouth disease virus.

The foot-and-mouth disease virus RNA polymerase complex was dissociated from cellular membranes with deoxycholate in the presence of dextran sulfate. The soluble polymerase complex was active in the cell-free synthesis of virus-specific RNA; solubilization of the complex permitted direct analysis of the cell-free reaction mixtures without recourse to RNA extraction. A major RNA-containing component found early during cell-free incubation ranged from approximately 140 to 300S. The final major products of the cell-free system were 37S virus RNA, 20S ribonuclease-resistant RNA, and a 50S component containing RNA.

Vaccination with leukemia cells expressing cell-surface-associated GM-CSF blocks leukemia induction in immunocompetent mice.

The fundamental basis for immunotherapy of leukemia is that leukemic cells express specific antigens that are not expressed by normal hematopoietic cells. However, the host immune system appears to be tolerant to leukemia cells. To overcome this tolerance, we vaccinated immunocompetent mice with murine leukemia cells (WEHI-3B and BCR-ABL+ 32D cells) transduced with a specifically constructed transmembrane form of granulocyte-macrophage colony-stimulating factor (tmGM-CSF). The transduced cells expressed tmGM-CSF on the cell-surface. To determine whether tmGM-CSF-expressing WEHI-3B leukemia cells would prevent leukemia formation as a vaccine, immunocompetent mice (BALB/c and C3H/HEJ) were immunized with lethally irradiated murine leukemia cells expressing cell-surface tmGM-CSF before challenging mice with murine leukemia cells. Two immunocompetent mouse models were investigated, either WEHI-3B cells in BALB/c mice or BCR-ABL+ 32D cells in C3H/HEJ mouse. The results showed that 100% of WEHI-3B/tmGM-CSF-vaccinated BALB/c mice and about 65% of 32D+ BCR-ABL/tmGM-CSF-vaccinated C3H/HEJ mice were protected from leukemia after leukemia cell challenge, whereas all non-vaccinated mice succumbed to leukemia. Spleen and marrow cell suspensions from vaccinated mice challenged with WEHI-3B cells lacked detectable GFP+ WEHI-3B cells at 82 days post-challenge. A significant delay of death was observed in C3H/HEJ mice challenged with the very aggressive DA-1 cell line expressing BCR-ABL. Vaccination of mice with WEHI-3B/CD40L cells protected 80% of the mice from the WEHI-3B challenge. Notably, 60% of the WEHI-3B/BALB/c mice were also protected from leukemia when WEHI-3B/tmGM-CSF vaccination was carried out after the leukemia challenge. In order to determine whether cellular immunity is involved in this vaccine-mediated protection, either CD4+ or CD8+ T cells were depleted from mice after the WEHI-3B/tmGM-CSF vaccination. The results indicate that CD8+ T-cells mediated the protective immune response provided by the irradiated tmGM-CSF-expressing WEHI-3B cells. In addition, vaccination of nude mice did not provide protection from WEHI-3B leukemia induction. Importantly, 80% of non-vaccinated mice were also protected from a WEHI-3B cell challenge after receiving spleen cells from vaccinated mice 1 day before challenge with leukemia cells. These results indicate that overexpression of tmGM-CSF on the leukemia cell-surface can enhance the recognition of leukemic cells by CD8+ T cells, and can either prevent or significantly delay leukemia induction. These findings suggest that injection of irradiated leukemia cells expressing cell-surface-bound GM-CSF has the potential as an immunological approach to treat leukemia.

Inhibition of v-Mos kinase activity by protein kinase A.

We investigated the effect of cyclic AMP-dependent protein kinase (PKA ) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37v-mos in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Combined tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies indicated that Ser-56 is the major in vivo phosphorylation site on v-Mos. In vivo phosphorylation at Ser-56 correlated with slower migration of the v-Mos protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, even though Ser-56 was phosphorylated by PKA, this phosphorylation was not involved in the inhibition of v-Mos kinase. The alanine-for-serine substitution at residue 56 did not affect the ability of v-Mos to autophosphorylate in vitro or, more importantly, to activate MEK1 in transformed NIH 3T3 cells. We identified Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. From our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation in mice could be explained at least in part by its inhibition of Mos kinase.

Inhibition of Bcr serine kinase by tyrosine phosphorylation.

The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

Detection of BCR-ABL proteins in blood cells of benign phase chronic myelogenous leukemia patients.

More than 95% of patients with chronic myelogenous leukemia (CML) contain an abnormal chromosome termed the Philadelphia chromosome (Ph1). Ph1 and the resulting BCR-ABL fused genes are markers for this type of leukemia. The product of the fused BCR-ABL genes is a protein of about 2000 amino acids termed P210 BCR-ABL. Although the BCR-ABL protein can be routinely detected in blood cells from blast crisis CML patients by assaying for its activated tyrosine kinase activity, detection of P210 BCR-ABL in early stage CML patients (chronic phase) has not yet been possible (S. A. Maxwell et al., Cancer Res., 47: 1731, 1987). A procedure involving Western blotting with an anti-ABL monoclonal antibody was developed that allows detection of P210 BCR-ABL and P145 ABL in cells from chronic phase and blast crisis CML patients, but as expected only P145 ABL was found in normal white blood cells. Most chronic phase patients also contained one to two ABL proteins with a molecular weight of about 190,000. Interestingly, the ratio of BCR-ABL to ABL proteins increased in four blast crisis patients compared to 18 chronic phase patients. Also, one chronic phase patient analyzed on three separate occasions lacked P210 BCR-ABL and exhibited only the Mr 190,000 form. This assay should also be useful in other leukemias that express altered forms of the ABL protein.

Sequences within the first exon of BCR inhibit the activated tyrosine kinases of c-Abl and the Bcr-Abl oncoprotein.

The Bcr-Abl oncoprotein is the primary causative factor in Philadelphia chromosome-associated leukemias. The activated tyrosine kinase of the Bcr-Abl oncoprotein is the primary driving force behind its oncogenic activity. We report here that a deleted form of Bcr [Bcr(64-413)], encompassing the Abl SH2 binding domains of Bcr, reduced the phosphotyrosine content of c-Abl and Bcr-Abl within cells and inhibited Bcr-Abl autophosphorylation activity in vitro. Similarly, a Bcr peptide phosphorylated on Ser-354 blocked the c-Abl and Bcr-Abl kinases in vitro, whereas the same peptide phosphorylated on Tyr-360 was not inhibitory. Bcr(64-413) was also resistant to tyrosine phosphorylation by either activated c-Abl or Bcr-Abl. Importantly, Bcr(64-413) interfered with the growth of Bcr-Abl-expressing cell lines. Our findings indicate that the Abl SH2 binding domain of Bcr in the phosphoserine form inhibits the Bcr-Abl oncoprotein but that tyrosine phosphorylation of this domain of Bcr reverses its inhibitory effects on Bcr-Abl. These results raise interesting questions about a possible role of Bcr or a Bcr-related molecule in modulating the activity of the Bcr-Abl oncoprotein and c-Abl itself.

P210 Bcr-Abl interacts with the interleukin 3 receptor beta(c) subunit and constitutively induces its tyrosine phosphorylation.

Chronic myelogenous leukemia is a neoplasm of pluripotent hematopoietic cells. The P210 Bcr-Abl oncoprotein is a deregulated cytoplasmic tyrosine kinase that has been shown to cause chronic myelogenous leukemia-like neoplasms in mice. Cytokines such as interleukin 3 and granulocyte/macrophage-colony-stimulating factor regulate the growth and differentiation of hematopoietic precursors. These cytokines activate two distinct signals to the nucleus. One signal is through the Ras pathway, and the second involves activation of Jak2. We demonstrated that Bcr-Abl co-immunoprecipitates with, and constitutively phosphorylates, the common beta(c) subunit of the interleukin 3 and granulocyte/macrophage-colony-stimulating factor receptors. Our data show that formation of this complex leads to the constitutive tyrosine phosphorylation of Jak2. It has been demonstrated that Bcr-Abl interacts with Grb2 and Shc, which in turn activates the Ras pathway. Our new findings raise the possibility that Bcr-Abl activates signaling through both pathways in a factor-independent fashion.

Analysis of P210bcr-abl tyrosine protein kinase activity in various subtypes of Philadelphia chromosome-positive cells from chronic myelogenous leukemia patients.

An altered c-abl gene product (P210bcr-abl) possessing associated tyrosine protein kinase activity was recently been reported in several blast chronic myelogenous leukemia (CML) cell lines. We have examined different morphological types of leukocytes directly obtained from patients at the blast crisis stage of CML for expression of P210bcr-abl tyrosine protein kinase activity. Phosphorylation of P210bcr-abl in an immune complex kinase assay using an anti-v-abl peptide serum was observed in blast cells from four Philadelphia chromosome (Ph1)-positive CML patients in blast crisis. P210bcr-abl protein kinase activity was detected regardless of whether the blast cells were of myeloid, lymphoid, or undifferentiated morphology. P210bcr-abl protein kinase activity was not detected in immune complexes either from leukocytes of four Ph1-negative CML patients in blast crisis, of five acute myelogenous leukemia patients, or in the promyelocytic cell line HL-60. Mature myeloid cells are associated with an inhibitory factor for not only P210bcr-abl protein kinase activity, but also protein kinases in general. Therefore, analyses of Ph1-positive benign phase CML myeloid cells, the majority of which are well differentiated, could not be successfully performed. The inhibition of P210bcr-abl protein kinase activity is not a specific property of mature cells from CML patients since granulocytes from a normal volunteer also demonstrated a similar effect. However, extracts of Ph1-positive cultured B-lymphocytes from a patient in benign phase demonstrated active P210bcr-abl protein indicating that the P210bcr-abl protein is expressed in an enzymatically active form in the earlier phases of CML. In addition to the previously reported P210 and P190 abl-related proteins, a novel Mr 53,000 protein was found to undergo phosphorylation at serine and tyrosine in immune complex kinase assays of two blast crisis CML cell lines (K562 and EM2) and in samples from blast crisis patients in which P210bcr-abl was detected. Peptide mapping by the Cleveland technique suggested that Mr 53,000 protein is unrelated to P210bcr-abl. Immune complex kinase assays of K562 cells with an anti-src serum (GD-11) yielded active c-src kinase and a Mr 50,000 phosphorylated protein, both of which were resistant to alkaline hydrolysis. Peptide mapping suggested that Mr 53,000 protein is related to Mr 50,000 protein which is precipitated with P210bcr-abl as an Mr 300,000 protein complex.

Expression of a truncated first exon BCR sequence in chronic myelogenous leukemia cells blocks cell growth and induces cell death.

We have shown that a deletion mutant form of Bcr [Bcr(64-413)] is a strong inhibitor of the tyrosine kinase of Bcr-Abl in vitro and also inhibits its oncogenic growth effects (Liu et al., Cancer Res., 56: 5120-5124, 1996). To determine the effects of this Bcr-Abl kinase inhibitor on chronic myelogenous leukemia (CML) cells, we cloned BCR(64-413) into a recombinant, replication-defective adenovirus to express useful quantities of Bcr(64-413) in a wide variety of cells in culture. Infection of Cos1 cells with plaque-purified virus at a multiplicity of infection of 20-40 induced high expression of Bcr(64-413) as detected by Western blotting. Infection of hematopoietic cells at modest multiplicities of infection (20-40) required special conditions involving shifting cycling cells to a nongrowing condition involving serum starvation and cell crowding. Under these conditions, both Bcr-Abl-positive and -negative hematopoietic cells can be efficiently infected by adenovirus, as demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of cells infected by beta-galactosidase (beta-GAL) adenovirus. We found that expression of Bcr(64-413) in Bcr-Abl-positive K562 and BV-173 cells, but not Bcr-Abl-negative SMS-SB cells, increased cell-cell clumping and inhibited cell growth. In contrast to the effects of the Bcr(64-413) adenovirus, the beta-GAL adenovirus, despite infecting both types of cells, did not block growth or increase cell-cell clumping of Bcr-Abl-positive and -negative hematopoietic cells. Expression of Bcr(64-413) protein in primary cultures of cells from CML patients with active disease interfered with cell growth, induced apoptosis (as measured by annexin staining), and increased cell-cell clumping, whereas the beta-GAL adenovirus and mock-infected cells lacked these effects. In contrast, normal marrow cells did not exhibit these effects on infection with Bcr(64-413) adenovirus. We conclude from these findings that Bcr(64-413) interferes with the oncogenic effects of Bcr-Abl and therefore has the potential for use in therapy of CML.

Requirement of two specific tyrosine residues for the catalytic activity of Bcr serine/threonine kinase.

Bcr is a novel serine/threonine protein kinase that is believed to require two cysteine pairs for activity (Maru and Witte, Cell, 67, 459, 1991). Tyrosine phosphorylated Bcr has dramatically reduced kinase activity, and tyrosine 360 of Bcr, which is one of the sites of phosphorylation by the Bcr-Abl oncoprotein, is required for transkinase activity (Liu et al., Mol. Cell Biol., 16, 998, 1996). Results presented here indicate that Bcr tyrosine 328 is also phosphorylated within Bcr-Abl expressing cells and is required for Bcr's serine/threonine kinase activity. Bcr Y328F, like Bcr Y360F, had defective transkinase activity but can autophosphorylate. However, the Y328F/Y360F double mutant of Bcr is defective in both trans- and autokinase activities. Taken together with the kinase inhibitory effects of tyrosine phosphorylation of Bcr by Bcr-Abl, our studies with tyrosine to phenylalanine Bcr mutants indicate that the hydroxyl residues of tyrosines 328 and 360 play crucial roles in Bcr's kinase activity.

Evidence for involvement of the protein kinase C pathway in the activation of p37v-mos protein kinase.

Protein kinases are known to undergo phosphorylation to regulate their activity. To determine whether the protein kinase activity of p37v-mos was similarly regulated, we investigated the influence of two well known protein kinases, namely protein kinase C and protein kinase A, on the activity of p37v-mos in vivo. NIH3T3 cells chronically transformed with Moloney murine sarcoma virus 124 were treated with high concentrations (200-400 nM) of phorbol 12-myristate 13-acetate (PMA) for 24-48 h, concentrations known to result in the total loss of protein kinase C by causing its translocation from the cytosol to cell membranes where it is downregulated. PMA treatment caused a drastic decrease in the protein kinase activity of p37v-mos without affecting its steady state level. Similar results were obtained with p85gag-mos expressed in ts110 Mo-MuSV transformed NRK cells. Control treatment with an inactive analogue of PMA, 4-alpha phorbol 12,13-didecanoate, had no effect on the p37v-mos protein kinase activity. Treatment of cells with a direct chemical inhibitor of protein kinase C, H-7 (1-(5-isoquinoline sulfonyl)-2-methylpiperazine dihydrochloride), approximately halved p37v-mos kinase activity, although the drug did not inhibit p37v-mos kinase activity directly in vitro. In contrast to the PMA effect, in vivo activation of protein kinase A by 8-(4-chlorophenylthio)-adenosine 3',5' cyclic monophosphate did not affect p37v-mos protein kinase activity levels. These findings indicate that the protein kinase C pathway but not the protein kinase A pathway modulates v-mos protein kinase activity.

P210 BCR-ABL is complexed to P160 BCR and ph-P53 proteins in K562 cells.

The BCR gene (Groffen et al., 1984) plays a critical role in the pathogenesis of human leukemias that involve the Philadelphia chromosome (Ph1) (Rowley, 1973; Nowell & Hungerford, 1960). Cells containing the Ph1 contain a chimeric gene formed from the fusion of BCR (Collins et al., 1987; Lifshitz et al. 1988) and ABL genes that results from the reciprocal translocation of segments of chromosomes 9 and 22 (Shtivelman et al., 1985). The product of this chimera is a 210 kDa protein, termed P210 BCR-ABL, that possesses an activated tyrosine kinase activity (Konopka et al., 1984; Kloetzer et al., 1985). Studies using long-term marrow culture systems and retrovirus-mediated gene transfer have documented that P210 BCR-ABL can stimulate the growth of immature hematopoietic precursor cell types (McLaughlin et al., 1987; Young & Witte, 1984). We have previously reported that P210 BCR-ABL exists in cytoplasmic complexes in association with a 53 kDa protein termed ph-P53 (Maxwell et al., 1987; Li et al. 1988). Similarly, BCR proteins have been found in cytoplasmic complexes containing ph-P53 in cells lacking the Ph1 (Li et al., 1989). These BCR protein complexes possess an associated ser/thr protein kinase activity. In this same study, we found that P210-containing complexes phosphorylate BCR proteins on tyrosine residues in vitro (Li et al., 1989). We now present results which demonstrate that P210 BCR-ABL is tightly associated with P160 BCR and ph-P53 proteins in cytoplasmic complexes from cells containing the Ph1.

Inhibition of c-mos protein kinase blocks mouse zygotes at the pronuclei stage.

The c-mos gene product is required for activation of the maturation-promoting factor (MPF) during oocyte maturation. The c-mos protein also acts as a cytostatic factor which is responsible for meiotic metaphase arrest of vertebrate eggs via stabilization of MPF. Here we show that mouse zygotes contain the c-mos protein. Introduction of a kinase-inhibitory anti-mos antibody into mouse zygotes 12 h after fertilization prevented the first cleavage of zygotes at the pronuclei stage. A second anti-mos antibody, known to allow the mos kinase to function, did not interfere with the formation of two-cell embryos. In addition to its known role in MPF activation in oocyte maturation and meiotic metaphase arrest, our findings indicate that the c-mos protein kinase is also required for completion of pronuclei breakdown following fertilization of mouse eggs.

Further characterization of the c-mos transcript and its cell cycle specific expression in NIH3T3 cells.

The mouse c-mos proto-oncogene is primarily expressed in germ cells. Our previous studies demonstrated c-mos RNA expression in mouse somatic cells, with the highest level present in the G2 phase of the cell cycle (Tsui et al., 1993). We have identified the transcription start site of this G2 specific c-mos transcript to be located about 1580 bp upstream from the open reading frame based on RT-PCR and RNase protection experiments. Upstream sequences containing this transcription start site directed highest expression of the luciferase reporter gene in M phase of the cell cycle. These results suggest that c-mos transcripts are produced in G2 phase and that c-Mos protein albeit at extremely low levels would accumulate in M phase.

Association of v-Mos with soluble vimentin in vitro and in transformed cells.

Our previous studies have shown that vimentin can serve as a substrate for the v-Mos protein kinase in vitro. Furthermore, the amount of vimentin molecules in Moloney murine sarcoma virus (Mo-MuSV) transformed cells is decreased relative to uninfected cells and a lower molecular weight form is observed in these v-mos transformed cells (Singh & Arlinghaus, Virology 173: 144-156, 1989). Vimentin filaments are hyperphosphorylated and disassemble when cells enter mitosis. Here, we show that vimentin was coprecipitated with p85gag-mos from mitotic cell extracts by two different anti-Mos antibodies that do not crossreact with vimentin. However, we were unable to detect vimentin/v-Mos complexes interphase extracts, possibly due to lack of soluble vimentin. p37env-mos was also found to associate with purified bovine lens vimentin in vitro. The significance of these findings with regard to v-mos-induced cellular transformation is discussed.

A novel 53 kDa protein complexed with P210bcr-abl in human chronic myelogenous leukemia cells.

Leukemic cells from patients with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) contain a 210 kDa protein (P210bcr-abl) with a protein tyrosine kinase activity that is a product of fused bcr and abl genes. We have prepared two monoclonal anti-peptide antibodies, one from each gene product, and have affinity purified each. Incubation of anti-abl (c-abl 51-64) immunoprecipitates of K562 cells with [gamma-32P]ATP in protein kinase assays resulted in the labeling of P210bcr-abl and a 53 kDa (ph-P53) protein. Increasing concentrations of antibody detected similar ratios of P210bcr-abl: ph-P53, suggesting the presence of a complex between the proteins. Several different anti-abl and anti-bcr antibodies detected the ph-P53/P210 complex. Sodium dodecyl sulfate (SDS) treatment without 2-mercaptoethanol eluted P210bcr-abl and ph-P53 from the monoclonal antibody in the form of complexes which migrated on 6% SDS-polyacrylamide gels and had apparent molecular weights of 275,000 and more than 500,000. Both complexes yielded ph-P53 and P210bcr-abl upon treatment with SDS-mercaptoethanol. Studies involving glycerol gradient centrifugation also detected complexes of P210bcr-abl and ph-P53. Our results indicate that ph-P53 is not a degraded product of P210bcr-abl, does not share antigenic determinants with P210bcr-abl since it is not recognized by anti-abl and bcr antibodies in immunoblots, is not the phosphorylated heavy chain of immunoglobulin G, and is different from p53 (the nonviral T protein) complexed to the large T antigen of simian virus 40. Previous studies (Maxwell et al., 1987) have shown that ph-P53 has a different peptide map than P210bcr-abl. Therefore, we conclude that ph-P53 is a distinct cellular protein complexed to P210bcr-abl in K562 cells.

Evidence for somatic cell expression of the c-mos protein [corrected].

The c-mos protein has been found to be enriched in germ cells of male mice, as described in a recent report from this laboratory (Herzog et al., Oncogene 3, 225, 1988). We report on further studies which indicate that the c-mos protein (a 41 to 43 kDa protein termed p43c-mos) is expressed in somatic tissues of mice and in cells grown in culture. In testes of mice, germ cell fractions have increased levels of p43c-mos relative to other cells of the testes. However, non-germ cells harbor significant levels of p43c-mos, as judged by comparison of testes from normal mice to those with mutations that affect the germ cell content of the testes. Thus, homozygous S1, at, and the W/Wv mutant mice are sterile due to severe deficiencies of germ cells. Such mice had only an estimated 50%-60% reduction in p43c-mos as judged by western immunoblotting using two different site-directed anti-mos antibodies. Similarly, X/X-sex reversed mice in which germ cells die after 10 days of age had only an 85% reduction of p43c-mos in mice 35 days of age. Thus, the germ cell content of testes did not correlated with p43c-mos levels in this tissue. Direct analyses of non-germ cells derived from mouse testes confirmed these findings, since Sertoli and Leydig cell lines grown in culture expressed p43c-mos. In addition, tissues such as kidney, liver, spleen and brain were found to contain p43c-mos. Surprisingly, mouse NIH3T3 cells were found to express significant levels of the c-mos protein based upon immunoblotting and one-dimensional peptide mapping experiments performed with both anti-mos antibodies. The concentration of the c-mos protein was not affected by expression of viral mos proteins. We conclude that the c-mos protein is enriched in male germ cells, but p43c-mos is also expressed in significant amounts in somatic tissues and in fibroblastic cells grown in culture.

Bcr phosphorylated on tyrosine 177 binds Grb2.

We and others have shown that the Bcr-Abl oncoprotein binds activators of the Ras pathway such as Grb2 and Shc. Grb2 binding is mediated through a phosphorylated tyrosine residue (Y177) located within a consensus Grb2 binding site encoded by the first exon of the BCR gene. Our results indicate that P160 BCR is tyrosine phosphorylated at the same site by Bcr-Abl in kinase assays (Puil et al., 1994). We performed experiments to determine whether Bcr, which was tyrosine phosphorylated within cells by activated c-Abl, could also bind Grb2, and whether phosphotyrosine 177 was the major binding site. Complexes between Bcr and Abl were detected in a hemopoietic cell line lacking Bcr-Abl and in COS1 cells coexpressing both Bcr and Abl proteins. P160 BCR was tyrosine phosphorylated in COS1 cells coexpressing Abl and Bcr proteins. Similarly, various deletion mutants of Bcr including BCRN553, BCRN413 and BCRN221 were tyrosine phosphorylated by activated c-Abl whereas BCRN159 was not. Wild-type Bcr and Bcr Y177F were examined under these conditions for their ability to co-precipitate with Grb2. The results showed that while wild-type tyrosine phosphorylated Bcr efficiently bound Grb2, tyrosine phosphorylated Bcr Y177F had greatly reduced Grb2-binding ability. Studies with GST-SH2 (Grb2) revealed that tyrosine phosphorylated Bcr was able to bind to GST SH2 (Grb2) but tyrosine phosphorylated Bcr Y177F was deficient in binding. These results indicate that the Bcr protein when phosphorylated at tyrosine 177 binds Grb2, thereby implicating Bcr as a potantial activator of the Ras pathway.

Requirement of the c-mos protein kinase for murine meiotic maturation.

Anti-sense mos oligonucleotides have been shown to block steps required for meiotic maturation of oocytes. In Xenopus oocytes, the block prevents germinal vesicle breakdown (GVBD), an early step in oocyte maturation. In mice the block induced by anti-sense mos occurs at a later step in oocyte maturation concerned with the first polar body emission. Here, we show that mouse oocyte maturation is blocked by introduction of anti-mos antibodies into immature functional oocytes. Antibodies that inhibit the mos kinase blocked GVBD. In contrast, anti-mos antibodies that permit the mos kinase to function do not block GVBD, but interfere with polar body formation. Antibodies pre-reacted with excess cognate peptide had no observable effects on maturation. These results indicate that the c-mos kinase function is required for activation of maturation-promoting factor (MPF) during meiosis. These findings are also consistent with our previous studies which show that the mos kinase directly phosphorylates cyclin B2, a component of MPF (Roy et al., 1990).