Heterogeneity in ERK activity as visualized by in vivo FRET imaging of mammary tumor cells developed in MMTV-Neu mice.

Human epidermal growth factor receptor2/Neu, which is overexpressed in about 30% of human breast cancers, transduces growth signals in large part via the Ras-Raf-MEK-ERK pathway. Nevertheless, it is a matter of controversy whether high ERK activity in breast cancer tissues correlates with better or worse prognosis, leaving the role of ERK activity in the progression of breast cancers unresolved. To address this issue, we live-imaged ERK activity in mammary tumors developed in mouse mammary tumor virus-Neu transgenic mice, which had been crossed with transgenic mice expressing a Förster resonance energy transfer biosensor for ERK. Observation of the tumor by two-photon microscopy revealed significant heterogeneity in ERK activity among the mammary tumor cells. The level of ERK activity in each cell was stable up to several hours, implying a robust mechanism that maintained the ERK activity within a limited range. By sorting the mammary tumor cells on the basis of their ERK activity, we found that ERK(high) cells less efficiently generated tumorspheres in vitro and tumors in vivo than did ERK(low) cells. In agreement with this finding, the expressions of the cancer stem cell markers CD49f, CD24 and CD61 were decreased in ERK(high) cells. These observations suggest that high ERK activity may suppress the self-renewal of mammary cancer stem cells.

Requirement of nef for HIV-1 infectivity is biased by the expression levels of Env in the virus-producing cells and CD4 in the target cells.

The nef gene of human immunodeficiency virus type 1 (HIV-1) encodes a 27 to 34 kDa myristoylated protein, which enhances viral infectivity in a single-round infection assay. The level of Nef enhancement of HIV-1 infectivity depends on the viral strains, on the target cells, and on the cells used for propagating the viruses. In this study, we aimed at clarifying the molecular basis of these differences in the requirement for Nef. We found that the requirement for Nef was increased when we decreased the quantity of Env protein in the virus-producing cells or the quantity of CD4 in the target cells. Both the wild-type and Nef-defective HIV-1 viruses were propagated in 293T cells, which did not express any CD4; therefore, Nef-induced CD4 down-regulation did not explain this phenomenon. Moreover, we did not observe any increase in the viral entry or fusion activity of gp120env in the wild-type HIV-1 compared to that in the Nef-defective HIV-1. Thus, we propose that Env on the virion and CD4 on the target cells have inhibitory effects on the post-entry step of the HIV-1 replication cycle, and that Nef functions to counteract this negative effect.

Lipid domains in the endocytic pathway.

Whereas endosomes connect with both exocytic and endocytic organelle via extensive lipid and protein traffic, each endosome has a distinct lipid and protein composition. Recent observations suggest that different lipid membrane domains exist even in the same endosome. These lipid domains, together with low pH milieu, may present a variety of micro-environments to cargo molecules. Evidence is accumulating which suggests that the alteration of these lipid microdomains may be involved in a number of pathological conditions.

Membrane recruitment of DOCK180 by binding to PtdIns(3,4,5)P3.

DOCK180 was originally identified as one of two major proteins bound to the Crk oncogene product and became an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. Further study has suggested that DOCK180 is involved in the activation of Rac by the CrkII-p130(Cas) complex. With the use of deletion mutants of DOCK180, we found that the C-terminal region containing a cluster of basic amino acids was required for binding to and activation of Rac. This region showed high amino-acid sequence similarity to the consensus sequence of the phosphoinositide-binding site; this led us to examine whether this basic region binds to phosphoinositides. For this purpose we used PtdIns(3,4,5)P(3)-APB beads, as reported previously [Shirai, Tanaka, Terada, Sawada, Shirai, Hashimoto, Nagata, Iwamatsu, Okawa, Li et al. (1998) Biochim. Biophys. Acta 1402, 292-302]. By using various competitors, we demonstrated the specific binding of DOCK180 to PtdIns(3,4,5)P(3). The expression of active phosphoinositide 3-kinase (PI-3K) did not enhance a DOCK180-induced increase in GTP-Rac; however, the expression of PI-3K translocated DOCK180 to the plasma membrane. Thus DOCK180 contained a phosphoinositide-binding domain, as did the other guanine nucleotide exchange factors with a Dbl homology domain, and was translocated to the plasma membrane on the activation of PI-3K.

The Nef protein of HIV-1 associates with rafts and primes T cells for activation.

The Nef protein is an important virulence factor of primate lentiviruses, yet the mechanisms by which it exerts this influence are imperfectly understood. Here, using an inducible system, we demonstrate that Nef increases IL-2 secretion from T cells stimulated via CD3 or CD28. This effect requires the conservation of the Nef myristoylation signal and SH3-binding proline-based motif. Together with several proteins involved in the initiation and propagation of T cell signaling, Nef associates with membrane microdomains known as rafts. The Nef-mediated superinduction of IL-2 reflects the activation of both NFAT and NFkappaB. Accordingly, Nef also enhances HIV-1 transcription in response to CD3 or CD28 stimulation. Nef-induced IL-2 hyperresponsiveness is also observed in primary CD4 lymphocytes. Overall, these data suggest that Nef acts at the level of rafts to prime T cells for activation. Likely consequences of this effect are the promotion of HIV-1 replication and the facilitation of virus spread.

Epidermal growth factor-dependent dissociation of CrkII proto-oncogene product from the epidermal growth factor receptor in human glioma cells.

Human glioma cells frequently overexpress epidermal growth factor receptor (EGFR). We found that the CrkII proto-oncogene product was associated with the EGFR in human glioma cells in the absence of epidermal growth factor (EGF). EGF stimulation of glioma cells induced the phosphorylation of tyrosine 221 of the CrkII protein, which correlates with its dissociation from the EGFR. By contrast, Shc and Grb2 were inducibly associated with the EGFR in response to EGF stimulation of glioma cells. In A431 cells, epidermoid carcinoma cells which overexpress EGFR, CrkII was tyrosine-phosphorylated and associated with the EGFR in an EGF-dependent manner. Therefore, the dissociation of CrkII from the EGFR upon stimulation with EGF appears to be specific to glioma cells. The Cbl oncogene product was also tyrosine-phosphorylated in U87MG glioma cells upon EGF stimulation. However, unlike in other cell lines, CrkII was not inducibly bound to Cbl in U87MG glioma cells. Thus, EGF-dependent binding of CrkII to phosphotyrosine-containing proteins appears to be suppressed in glioma cells. To evaluate the physiological role of dissociation of CrkII from EGFR, we expressed the CrkII-23 mutant in glioma cells. CrkII-23 mutant, which was isolated as a suppressor gene of the EGF-dependent transformation of NRK cells, binds constitutively to EGFR. We found that expression of CrkII-23 inhibited the anchorage-independent growth of the glioma cells in the presence of EGF. Taken together, these data implicate EGF-dependent dissociation of CrkII from EGFR in the oncogenicity of human glioma cells.

Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i)

Many receptors for neuropeptides and hormones are coupled with the heterotrimeric G(i) protein, which activates the p42/44 mitogen-activated protein kinase (ERK/MAPK) cascade through both the alpha- and betagamma-subunits of G(i). The betagamma-subunit activates the ERK/MAPK cascade through tyrosine kinase. Constitutively active G(alpha)i2 (gip2) isolated from adrenal and ovarian tumours transforms Rat-1 fibroblasts and also activates the ERK/MAPK cascade by an unknown mechanism. The ERK/MAPK pathway is activated by Ras, and is inhibited when the low-molecular-mass GTP-binding protein Rap1 antagonizes Ras function. Here we show that a novel isoform of Rapl GTPase-activating protein, called rap1GAPII, binds specifically to the alpha-subunits of the G(i) family of heterotrimeric G-proteins. Stimulation of the G(i)-coupled m2-muscarinic receptor translocates rap1GAPII from the cytosol to the membrane and decreases the amount of GTP-bound Rap1. This decrease in GTP-bound Rap1 activates ERK/MAPK. Thus, the alpha-subunit of G(i) activates the Ras-ERK/MAPK mitogenic pathway by membrane recruitment of rap1GAPII and reduction of GTP-bound Rap1.

Activation of Rac1 by a Crk SH3-binding protein, DOCK180.

DOCK180 is involved in integrin signaling through CrkII-p130(Cas) complexes. We have studied the involvement of DOCK180 in Rac1 signaling cascades. DOCK180 activated JNK in a manner dependent on Rac1, Cdc42Hs, and SEK, and overexpression of DOCK180 increased the amount of GTP-bound Rac1 in 293T cells. Coexpression of CrkII and p130(Cas) enhanced this DOCK180-dependent activation of Rac1. Furthermore, we observed direct binding of DOCK180 to Rac1, but not to RhoA or Cdc42Hs. Dominant-negative Rac1 suppressed DOCK180-induced membrane spreading. These results strongly suggest that DOCK180 is a novel activator of Rac1 and involved in integrin signaling.

Evidence that DOCK180 up-regulates signals from the CrkII-p130(Cas) complex.

DOCK180 is one of the two principal proteins bound to the SH3 domain of the adaptor protein CrkII. Here, we have studied the involvement of DOCK180 in integrin signaling. DOCK180 was neither phosphorylated nor bound to CrkII in quiescent NIH 3T3 cells and 3Y1 cells. We found that DOCK180 was phosphorylated and bound to CrkII in NIH 3T3 cells stimulated with integrin and also in 3Y1 cells transformed by v-src or v-crk. The binding of DOCK180 to CrkII correlated with the binding of CrkII to p130(Cas), which is a major CrkII SH2 domain-binding protein at focal adhesions. In a reconstitution experiment, expression of DOCK180 induced hyperphosphorylation of p130(Cas) and a concomitant increase in the amount of CrkII bound to p130(Cas). Similarly, binding of DOCK180 to CrkII was also enhanced by the coexpression of p130(Cas). Finally, we found that coexpression of p130(Cas) and CrkII with DOCK180 induced local membrane spreading and accumulation of DOCK180-CrkII-p130(Cas) complexes at focal adhesions. These findings suggest that DOCK180 positively regulates signaling from integrins to CrkII-p130(Cas) complexes at focal adhesions.

Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth factor receptor.

CrkII adaptor protein becomes tyrosine-phosphorylated upon various types of stimulation. We examined whether tyrosine 221, which has been shown to be phosphorylated by c-Abl, was phosphorylated also by other tyrosine kinases, such as epidermal growth factor (EGF) receptor. For this purpose, we developed an antibody that specifically recognizes Tyr221-phosphorylated CrkII, and we demonstrated that CrkII was phosphorylated on Tyr221 upon EGF stimulation. When NRK cells were stimulated with EGF, the tyrosine-phosphorylated CrkII was detected at the periphery of the cells, where ruffling is prominent, suggesting that signaling to CrkII may be involved in EGF-dependent cytoskeletal reorganization. The EGF-dependent phosphorylation of CrkII was also detected in a c-Abl-deficient cell line. Moreover, recombinant CrkII protein was phosphorylated in vitro by EGF receptor. These results strongly suggest that EGF receptor directly phosphorylates CrkII. Mutational analysis revealed that the src homology 2 domain was essential for the phosphorylation of CrkII by EGF receptor but not by c-Abl, arguing that these kinases phosphorylate CrkII by different phosphorylation mechanisms. Finally, we found that the CrkII protein phosphorylated upon EGF stimulation did not bind to the phosphotyrosine-containing peptide and that CrkII initiated dissociation from EGF receptor within 3 min even with the sustained tyrosine phosphorylation of EGF receptor. This result implicated phosphorylation of Tyr221 in the negative regulation of the src homology 2-mediated binding of CrkII to EGF receptor.

Inhibition of human immunodeficiency virus type 1 virion entry by dominant-negative Hck.

To study the role of Src family tyrosine kinases in infection with human immunodeficiency virus type 1 (HIV-1), we constructed an Hck mutant, HckN, that hinders signaling from wild-type Hck. HIV-1 produced in HckN-expressing cells was significantly less infectious to HeLa-CD4-LTR-beta-gal (MAGI) cells than HIV-1 produced in mock-transfected cells. The inhibitory effect of HckN was compensated for by the expression of vesicular stomatitis virus G protein. Finally, we found that the HIV-1 produced in the HckN-expressing cells entered into the cells less efficiently than did the control HIV-1. These results suggest that the Src family tyrosine kinases regulate entry of HIV-1 into target cells.

Expression of high-mobility group-1 mRNA in human gastrointestinal adenocarcinoma and corresponding non-cancerous mucosa.

An 1194-nucleotide complementary DNA clone, FM1, encoding a human high-mobility group-1 protein (HMG-1) was isolated from a well-differentiated human gastric-carcinoma cell line complementary DNA library by a differential screening method. FM1 is similar to the published human HMG-1 in mature protein, with only 3 different codons at positions 11, 149, and 190. We analyzed 33 gastric and colorectal adenocarcinomas for expression of the FM1 gene. Northern-blot analysis revealed that all of the cancers expressed FM1 at a higher level than in corresponding non-cancerous mucosa, with 2 transcripts of approximately 1.4 and 2.4 kilobases. The FM1 expression level in the non-cancerous tissues increased with the depth of accompanying cancer invasion. Only 18.2% of well-differentiated cancers showed a higher expression level in corresponding non-cancerous tissues, whereas the expression in corresponding non-cancerous tissues was significantly higher in moderately (60%) and poorly differentiated (83.3%) cancers. In situ hybridization demonstrated the location of FM1 mRNA in well- and poorly differentiated gastric-cancer cells as well as in non-cancerous tissue adjacent to poorly differentiated gastric cancer, but no hybridization was detected in normal epithelial cells adjacent to well-differentiated gastric cancer. These findings may provide new information on HMG-1 mRNA expression in human gastrointestinal cancer and suggest a correlation between FM1 mRNA expression to the differentiation and the stage of human gastrointestinal adenocarcinomas.

Bilateral breast tumors, malignant phyllodes tumor and invasive lobular carcinoma in a 46,XX/46,XY mosaic female with family history of breast cancer.

Bilateral breast tumors, a malignant phyllodes tumor in the right breast and an invasive lobular carcinoma in the left breast, occurred in a 47-year-old woman with 46XX/46XY mosaic karyotype in her peripheral blood lymphocytes and intersex external genitalia. Postmortem examination revealed bilateral ovotestis. Three of the patient's sisters also had breast cancer. In situ hybridization with a Y-specific probe revealed Y-chromosome-specific signal in both tumors, suggesting that the clonal origin of tumors in this patient was Y-containing cells. Androgen-receptor polymorphism also revealed a monoallelic X chromosome pattern in the recurrent phyllodes tumor tissue taken at autopsy, in addition to loss of heterozygosity demonstrated at locus TP53. The slippage of the CA repeats in the tumor was also shown at the loci of D5S82 and D11S527. The mechanistic basis for the occurrence of bilateral malignant tumors of the breast, XX/XY mosaicism, and familial clustering of breast cancer is still unknown. The present study, however, suggests that the sex chromosome abnormality may have modified the cancer phenotype in a manner similar to breast cancer in Klinefelter's syndrome (though phenotypically male) and the Y chromosome may have promoted cell growth.

Role of Crk oncogene product in physiologic signaling.

v-Crk is a member of the family of adaptor-type signaling molecules that consist mostly of SH2 and SH3 domains. The cellular homologs of v-Crk includes CrkI, CrkII, and CrkL; these have been isolated from species ranging from lower vertebrates to man. Crk-family proteins are involved in a variety of signaling cascades such as those of growth factor receptor, integrin, T-cell receptor, B-cell antigen receptor, and cytokines. It has been postulated that the primary function of Crk is to recruit cytoplasmic proteins in the vicinity of tyrosine kinases through SH2-phosphotyrosine interaction. Thus, the output from Crk depends on the SH3-binding proteins, which include the C3G guanine nucleotide exchange protein for Rap1, Abl tyrosine kinase, DOCK180, and the Sos guanine nucleotide exchange protein for Ras. The variety of the Crk-binding proteins indicate the pleiotropic function of Crk.

DOCK180, a major CRK-binding protein, alters cell morphology upon translocation to the cell membrane.

CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Western blotting with CRK SH3 has demonstrated that it binds to 135- to 145-, 160-, and 180-kDa proteins. The 135- to 145-kDa protein is C3G, a CRK SH3-binding guanine nucleotide exchange protein. Here, we report on the molecular cloning of the 180-kDa protein, which is designated DOCK180 (180-kDa protein downstream of CRK). The isolated cDNA contains a 5,598-bp open reading frame encoding an 1,866-amino-acid protein. The deduced amino acid sequence did not reveal any significant homology to known proteins, except that an SH3 domain was identified at its amino terminus. To examine the function of DOCK180, a Ki-Ras farnesylation signal was fused to the carboxyl terminus of DOCK180, a strategy that has been employed successfully for activation of adaptor-binding proteins in vivo. Whereas wild-type DOCK180 accumulated diffusely in the cytoplasm and did not have any effect on cell morphology, farnesylated DOCK180 was localized on the cytoplasmic membrane and changed spindle 3T3 cells to flat, polygonal cells. These results suggest that DOCK180 is a new effector molecule which transduces signals from tyrosine kinases through the CRK adaptor protein.

CYP1A1 and CYP2E1 polymorphism and lung cancer, case-control study in Rio de Janeiro, Brazil.

Msp I polymorphism and exon 7 Ile-Val polymorphism of CYP1A1, and Rsa I polymorphism of CYP2E1 were studied in lung cancer patients and controls in Rio de Janeiro, Brazil. Of the three polymorphisms studied, only the exon 7 polymorphism of CYP1A1 (Val-containing genotypes) had a distribution which was statistically significant in the patients and controls. The contribution of Val containing genotypes of CYP1A1 exon 7 was greater in the subpopulation of squamous cell carcinoma patients with a lower life-time smoking consumption (OR, 2.92 vs 1.97). This association is consistent with the previous findings by Kawajiri et al. and the first observation of the positive association of this locus with lung cancer in a Western population (Kawajiri K, Nakachi K, Imai K, Yoshii A, Shimada N, Watanabe J. FEBS Let 1990; 263, 131-133). Furthermore, together with the lack of association of Msp I polymorphism in the non-coding region of CYP1A1, the locus truly responsible for lung cancer risk among pleural polymorphisms of CYP1A1 appeared to be exon 7 Ile-Val polymorphism. In the future, investigations of multiple markers in different ethnic populations may reveal cancer risk markers common to all mankind.

The heme-binding region polymorphism of cytochrome P450IA1 (CypIA1), rather than the RsaI polymorphism of IIE1 (CypIIE1), is associated with lung cancer in Rio de Janeiro.

Ile-Val polymorphism in exon 7 of cytochrome P450IA1 (CypIA1) and RsaI polymorphism of cytochrome P450IIE1 (CypIIE1) were examined in a case-control study of lung cancer in Rio de Janeiro, Brazil. The Val-containing genotype in exon 7 of CypIA1 was found to be associated with lung cancer in this population (odds ratio, 2.26; 95% confidence interval, 1.14-4.47 for 99 cases versus 108 controls of 123 matched pairs), whereas RsaI polymorphism in CypIIE1 was not associated with lung cancer susceptibility. In squamous cell carcinoma, the degree of association of Val-containing genotype was greater in those with fewer pack-years of smoking. The RsaI polymorphism of CypIIE1 has a different distribution from the Japanese pattern and is not associated with lung cancer. When we analyzed the association of Ile-Val polymorphism to MspI polymorphism of CypIA1, the Val/Val homozygote was found only in the subpopulation with the MspI site-present homozygote. The apparent lack of association of CypIA1 MspI polymorphism with lung cancer in this area reported in our previous study and the results of the present study indicate that the "true" responsible site for lung cancer susceptibility should be the Ile-Val polymorphism in the catalytic site of CypIA1.

Overexpression of ERK, an EPH family receptor protein tyrosine kinase, in various human tumors.

The ERK gene has been isolated as a genomic DNA encoding a part of the receptor protein-tyrosine kinase which belongs to the EPH subfamily. We previously identified a partial complementary DNA (cDNA) encompassing the catalytic domain of ERK from the expression library of human gastric cancer with an antiphosphotyrosine antibody. Using this cDNA as a probe, the cDNAs encoding mature ERK protein were isolated. The putative mature ERK protein, a total of 967 deduced amino acid residues, showed high homology with chicken Cek5 (92.5%) and mouse Nuk (99.1%). Chromosomal in situ hybridization revealed that human ERK cDNA is localized to chromosome 1p34-35. In Northern blot analysis of normal human tissues, the ERK gene was ubiquitously expressed mainly in cells of epithelial origin but not in the brain. Studies on RNAs from 76 human tumor tissues and cell lines showed that ERK is expressed at higher levels in various tumors of epithelial origin than in corresponding normal tissues, most frequently in gastric cancers (12 of 16, 75.0%). Overexpression of ERK was also detected in one osteosarcoma cell line. These findings suggest that ERK plays some significant role in carcinogenesis in the stomach and other tissues.