BACKGROUND: Cytotoxic function of killer cells is inhibited by specific recognition of class I MHC molecules on target cells by inhibitory killer Ig-like receptors (KIR) expressed on NK cells and some cytotoxic T cells. The inhibitory effect of KIR is accomplished by recruitment of SH2-containing protein tyrosine phosphatase (SHP) to the phosphotyrosine residues in the cytoplasmic tail. METHODS: By in vitro coprecipitation experiments and transfection analysis, we investigated the association of KIR with an adaptor protein Shc in Jurkat T cells. RESULTS: The cytoplasmic tail of KIR appeared to associate with an adaptor protein Shc in Jurkat T cell lysates. Similar in vitro experiments showed that phosphorylated KIR cytoplasmic tail bound SHP-1 and Shc in Jurkat T cell lysates. The association of KIR with Shc was further confirmed by transfection analysis in 293T cells. Interestingly, however, Shc appeared to be replaced by SHP-2 upon engagement of KIR in 293T cells. CONCLUSION: Our data indicate that KIR associate with an adaptor protein Shc in Jurkat T cells, and suggest that KIR might have an additional role which is mediated by this adaptor protein.
Cell Proliferation ; Cytoplasm ; Killer Cells, Natural ; Phosphotyrosine ; Protein Tyrosine Phosphatases ; T-Lymphocytes ; Transfection

PURPOSE: Anti-p185HER2/neu monoclonal antibody (mAb) that can induce phenotypic reversion and monoclonal antibodies specific for the p185HER2/neu growth factor receptor that are able to diminish its kinase-signaling properties represent a specific advance in the therapy for p185HER2/neu-expressing human cancers. With mAb treatment, down-regulation of p185HER2/neu surface receptors and attenuation of the kinase-signaling properties have been observed and regarded as a basic phenomenon; however, the mechanisms for mAb-induced phenotypic reversion are not clear. METHODS: We used human tumor-cell lines of SK-BR-3, T6-17, and U373MG. With immunoprecipitation and Western blotting, we investigated the changes in p185HER2/neu receptor phosphorylation and the expression of signal-regulatory proteins (SIRPs) after mAb treatment. To identify the proteins interacting with Tat-binding protein-1 (TBP1), we used the Clonotech Gal4 matchmaker two-hybrid system. RESULTS: Minimal to moderate reduction in phosphotyrosine (pTyr) content was observed in SK-BR-3 and T6-17 cells with short-term (10-30 minutes) incubation after mAb treatment, but that did not alter total p185HER2/neu receptor density. SIRPs phosphorylation after peptide treatment was increased. With mAb treatment, three proteins were shown to interact with TBP1, and all of the interacting proteins are subunits of proteasome 26S. Collectively, anti- p185HER2/neu mAb or peptide down-regulates the surface receptors and attenuates the kinase signaling, which then both induces higher proteasome activity through increased TBP1 and increases SIRPs expres sion. CONCLUSION: Increased proteasomal activity may degrade abnormal proteins and increased SIRPs may regulate signal transduction toward the norm. Therefore, activation of a protein-degradation pathway and induction of signal-regulatory proteins may be possible mechanisms for the ultimate anti-tumor effects of the anti-p185HER2/neu mAb or peptide.
Antibodies, Monoclonal ; Blotting, Western ; Down-Regulation ; Humans ; Immunoprecipitation ; Phosphorylation ; Phosphotransferases ; Phosphotyrosine ; Proteasome Endopeptidase Complex ; Signal Transduction

Resistance to the induction of apoptosis is a possible mechanism by which tumor cells can survive anti-neoplastic treatments. Melanoma is notoriously resistant to anti-neoplastic therapy. Previous studies have demonstrated focal adhesion kinase (FAK) overexpression in melanoma cell lines. Given its probable role in mediating resistance to apoptosis, many researchers have sought to determine whether the downregulation of FAK in melanoma cells would confer a greater sensitivity to anti-neoplastic agents. Genistein is a known inhibitor of protein-tyrosine kinase (PTK), which may attenuate the growth of cancer cells by inhibiting the PTK-mediated signaling pathway. This present study was undertaken to investigate the effect of genistein on the expression of FAK and cell cycle related proteins in the G361 melanoma cell line. Genistein was found to have a preferential cytotoxic effect on G361 melanoma cells over HaCaT normal keratinocytes. Genistein decreased the expression of 125 kDa phosphotyrosine kinase and the FAK protein in particular. Genistein treatment did not affect the expression of p53 in G361 cells in which p21 is upregulated. The expression of cyclin B and cdc2 was downregulated by genistein treatment. Taken together, our data indicate that genistein induces the decreased proliferation of G361 melanoma cells via the inhibition of FAK expression and regulation of cell cycle genes. This suggests that the use of genistein may be a viable approach to future melanoma treatments.
Apoptosis ; Cell Cycle ; Cell Line ; Cyclin B ; Down-Regulation ; Focal Adhesion Protein-Tyrosine Kinases ; Focal Adhesions ; Genes, cdc ; Genistein ; Keratinocytes ; Melanoma ; Negotiating ; Phosphotransferases ; Phosphotyrosine ; Protein-Tyrosine Kinases ; Proteins

The objective of this study is to determine if the elevated retinal levels of phosphotyrosine in the Zucker diabetic fatty rats can be reduced by systemic administration of genistein,a protein tyrosine kinase inhibitor. Sixteen male Zucker diabetic fatty rats were compared with sixteen malelean litter mates as a control. Rats were injected intraperitoneally with 13.6mg/kg B.Wt.of genistein (treated group) or dimethyl sulfoxide (DMSO)(non-treated group) twice two hours apart. Twenty four hours later, the retinas were processed for western blot analysis and immunohistochemical study. The Zucker diabetic fatty rats were found to have increased levels of phosphotyrosine as compared with the lean controls. Intraperitoneal administration of genistein strongly inhibited protein tyrosine phosphorylation in the diabetic rats.Genistein also inhibited the increase in PCNA,but this was less obvious than the decrease in phosphotyrosine. In the diabetic rats, the levels of activated PI3-K and MAPK were increased over control rats. Genistein reduced the levels of PI3-K and MAPK. Immunohistochemical studies of the retina showed decreased immunopositive reaction of PCNA in the retina of the diabetic rats treated with genistein. In conclusion, elevat-ed levels of phospotyrosine in the Zucker diabetic rats can be reduced by systemic administration of genistein. Genistein also reduced, although less, the levels of PCNA and activated forms of PI3-K and MAPK. Our results suggest that PTK inhibitor like genistein may find a role in the reduction of proliferative complications of diabetes mellitus.
Animals ; Blotting, Western ; Diabetes Complications ; Dimethyl Sulfoxide ; Genistein* ; Humans ; Immunohistochemistry ; Male ; Phosphorylation ; Phosphotyrosine ; Proliferating Cell Nuclear Antigen ; Protein-Tyrosine Kinases ; Rats* ; Retina ; Retinaldehyde ; Tyrosine

OBJECTIVESTo compare expressions of tyrosine-phosphorylated proteins in different hepatocellular carcinoma cell lines with different metastasis potential and to screen key molecules associated with HCC metastasis and recurrence.

METHODSUsing two-dimensional electrophoresis, Western blotting and MALDI-TOF-MS/MS, we analyzed tyrosine-phosphorylated protein profiles of Hep3B, MHCC97L and MHCC97H, HCC cell lines with different metastasis potentials.

RESULTS10 spots were detected in Hep3B, 19 in MHCC97L and 17 in MHCC97H. Seventeen significantly different phosphotyrosine proteins in gel were identified by MALDI-TOF-MS/MS, including Annexin I.

CONCLUSIONThe changed expression of tyrosine-phosphorylated proteins is associated with HCC metastasis and recurrence.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Neoplasm Metastasis ; Neoplasm Proteins ; analysis ; Phosphotyrosine ; analysis

One mechanism of vascular damage in diabetic retinopathy is thought to result from the effect of hyperglycemia on the neural retina and microvasculature eventually causing the signs of capillary closure and clinical retinopathy. We reasoned that protein tyrosine kinase (PTK) pathways are abnormally activated in the retina prior to the onset of clinical changes. In this study, twelve Zucker diabetic fatty rats were compared with twelve male lean litter mates as control. The rats were examined for changes in the relative retinal levels of angiogenic growth factors, phosphotyrosine, PCNA, and tyrosine kinase pathway intermediates by western blot analysis. We found that the Zucker diabetic fatty rats have increased levels of the angiogenic growth factors, VEGF and PDGF. The levels of phosphotyrosine and PCNA, and phosphorylated intermediates, MAPK and PI3-K were also increased as compared with the lean control rats. These data indicate that the microvascular changes known to occur in this model are associated with elevation of VEGF and PDGF prior to the development of clinical apparent retinopathy. The elevations of VEGF and PDGF may explain in part the activation of the tyrosine kinase pathways, especially pathways that include MAPK and PI3K.
Animals ; Blotting, Western ; Capillaries ; Diabetic Retinopathy ; Humans ; Hyperglycemia ; Intercellular Signaling Peptides and Proteins ; Male ; Microvessels ; Phosphotyrosine ; Proliferating Cell Nuclear Antigen ; Protein-Tyrosine Kinases* ; Rats* ; Retina ; Retinaldehyde ; Vascular Endothelial Growth Factor A

We investigated the co-stimulatory role of a cell-surface protein, CD99. Co-ligation of CD99 and suboptimal CD3 induced T-cell activation to a level comparable to that obtained with optimal CD3 or CD3+CD28. We also noted concomitant enhancement of the earliest T-cell receptor (TCR) signaling events. In addition, co-ligation of CD99 and CD3 led to translocation of TCR complexes into the lipid raft, without concomitant migration of CD99 to the raft, and consequent enhancement of TCR zeta-mediated signal 1. These data demonstrate the unique properties of CD99 co-stimulation that distinguish this molecule from CD28 and other raft-resident co-stimulatory factors.
Antigens, CD/*immunology ; Antigens, CD3/immunology ; Cell Adhesion Molecules/*immunology ; Down-Regulation ; Humans ; Jurkat Cells ; Lymphocyte Activation/*immunology ; Membrane Microdomains/*immunology ; Membrane Proteins/*immunology ; Phosphorylation ; Phosphotyrosine/*metabolism ; Protein Transport ; Receptors, Antigen, T-Cell/*immunology ; T-Lymphocytes/*immunology

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein of unknown function in vivo, is abundantly expressed in myelinating glia in two isoforms, CNP1 and CNP2. In this study, immunoblot analysis showed that CNP1 is the major isoform in adult forebrain, and that both isoforms are included in the postsynaptic density (PSD) fraction and tyrosine-phosphorylated at the basal level. However, subcellular distribution and detergent extraction data showed that CNP is nonspecifically associated with the PSD fraction. Immunocytochemistry revealed that CNP is detected, in a weak but punctate pattern, in dissociated rat hippocampal neurons of 3 days to 2 weeks in vitro. The CNP-positive punctae were distributed throughout soma and dendrites, and distinct from PSD95-positive ones. Immunoblot analysis indicated that CNP is also expressed in neuronal stem cell lines, HiB5 and F11. Interestingly, in addition to the known two isoforms, a new CNP isoform of MW 45 kDa was expressed in these cell lines and was the major type of isoform in F11 cells. Taken together, our data suggest that CNP is expressed in the early stage of in vitro development and nonspecifically included in the adult rat PSD fraction.
2',3'-Cyclic-Nucleotide Phosphodiesterases/*metabolism ; Aging/physiology ; Animals ; Cells, Cultured ; Hippocampus/cytology/metabolism ; Immunohistochemistry ; Nerve Tissue Proteins/*metabolism ; Neurons/metabolism ; Phosphotyrosine/metabolism ; Prosencephalon/cytology/*metabolism ; Rats ; Rats, Sprague-Dawley ; Substrate Specificity

PURPOSE: Hydrogen peroxide has been implicated as a causative factor of various cellular dysfunction, cell death, transformation. In addition, oxidative stress has been suggested as a crucial inducer of cataract formation not only the nuclear-type cataract but also anterior polar type cataract. Transformation of lens epithelial cells accompanying accumulation of extracellular matrix molecules, and cell migration is observed in the cataract forming area of lens. In the present study, we investigated in the migration of lens epithelial cells and the activation of focal adhesion kinase(FAK), because there have been many reports showing that activation of FAK increased cell migration. METHOD: After treatment hygrogen peroxide in a dose-dependent on HLE B-3 cell line, we have performed immuno fluorenscence staing of actin stress fiber and migration assay, and then isolated total RNA to identify expression of MMP-2 from the cell line. To examine FAK activity, we are performed Phosphotyrosine Immunoblot analysis. RESULT: We observed the increased cell migration in response to hydrogen peroxide in a dose dependent manner. In addition, we observed that the phosphorylation of focal adhesion kinase was increased with the treatment of hydrogen peroxide. Also, we examined the expression of the matrix metalloproteinases-2(MMP-2) which disintegrates extracellular matrix and participates in cell migration. The result showed that the mRNA level of MMP-2 did not increase by hydrogen peroxide. CONCLUSIONS: The results suggest that hydrogen peroxide enhanced migration of lens epithelial cell, and that FAK may play a role in the process of cell migration. In conclusion, migration of lens epithelial cell and phosphorylation of focal adhesion kinase was increased by treatment of hydrogen peroxide. Thus oxidative stress plays a crucial role in the transformation of lens epithelial cells and anterior polar type cataract formation.
Actins ; Cataract ; Cell Death ; Cell Line ; Cell Movement ; Epithelial Cells* ; Extracellular Matrix ; Focal Adhesion Protein-Tyrosine Kinases ; Focal Adhesions ; Hydrogen Peroxide* ; Hydrogen* ; Oxidative Stress ; Phosphorylation ; Phosphotyrosine ; RNA ; RNA, Messenger ; Stress Fibers

Lung cancer is a deadly disease that is difficult to diagnose and even more difficult to treat effectively. Many pathways are known to affect tumor growth, and targeting these pathways provides the cornerstone by which cancer is treated. Somatostatin receptors (SSTR) are a family of G protein coupled receptors that signal to alter hormonal secretion, increase apoptosis, and decrease cellular proliferation. These receptors are expressed in many normal and malignant cells, including both small cell and non-small cell lung cancer. Synthetic analogs of SSTRs are commercially available, but their effects in lung cancer are still largely uncertain. Signaling pathway studies have shown that SSTRs signal through phosphotyrosine phosphatases to induce apoptosis as well as to decrease cell proliferation. Radiolabeled SSTR2 analogs are utilized for radiographic imaging of tumors, which, when combined with positron emission tomography-computed tomography (PET-CT) may improve detection of lung cancer. These radiolabeled SSTR2 analogs also hold promise for targeted chemotherapy as well as radiotherapy. In this review, we summarize what is known about SSTRs and focus our discussion on the knowledge as it relates to lung cancer biology, as well as discuss current and future uses of these receptors for imaging and therapy of lung cancer.
Apoptosis ; Biology ; Carcinoma, Non-Small-Cell Lung ; Cell Proliferation ; Electrons ; Humans ; Lung ; Lung Neoplasms ; Molecular Imaging ; Neuroendocrine Tumors ; Phosphoric Monoester Hydrolases ; Phosphotyrosine ; Receptors, G-Protein-Coupled ; Receptors, Somatostatin ; Somatostatin