Biomedical subjects

Z J Zhao

Publications and source records attributed to Z J Zhao.

At least 19 recordsLinked to original sources

PubMed2001

Association of tyrosine phosphatase SHP-2 with F-actin at low cell densities.

SHP-2 is an intracellular SH2 domain-containing protein-tyrosine phosphatase with an essential role in cell signaling. Here we demonstrate that localization of SHP-2 is regulated by cell density in a cell adhesion-dependent manner. When cells were plated at low densities, SHP-2 was distributed in Triton X-100-insoluble fractions, whereas it was totally soluble when cells were plated at high densities or when low density cells approached confluency. In all cases, the total protein level of SHP-2 was not changed. Fluorescent cell staining revealed that SHP-2 was co-localized with actin stress fibers to the cell peripheral at low cell densities but was diffused in the entire cytoplasm at high cell densities. Transient transfection of cells with truncated forms of SHP-2 demonstrated that the catalytic domain of the enzyme was responsible for the density-regulated distribution of SHP-2, but the catalytic activity was not required. An in vitro co-sedimentation study demonstrated direct binding of full-length and SH2 domain-truncated forms of SHP-2 to F-actin. The data indicate that SHP-2 is regulated by cell density and that it may have a role in assembling and disassembling of the actin network.

3T3 Cells↗

PubMed2001

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.

Oligopeptides↗

PubMed2001

FYVE-DSP2, a FYVE domain-containing dual specificity protein phosphatase that dephosphorylates phosphotidylinositol 3-phosphate.

We have recently isolated FYVE-DSP1, a FYVE domain-containing dual specificity protein phosphatase (R. Zhao, Y. Qi, and Z. J. Zhao, Biochem. Biophys. Res. Commun. 270, 222--229 (2000)). Here, we report a novel isozyme that we designated FYVE-DSP2. FYVE-2 contains a single FYVE domain at the C-terminus, and it shares approximately 47% overall sequence identity with FYBE-DSP1. Genomic sequence analyses revealed that the FYVE-DSP1 and FYVE-DSP2 genes share similar intron/exon organization. They are localizedon human chromosome 22q12 and chromosome 17, respectively. Like FYVE-DSP1, recombinant FYVE-DSP2 dephosphorylated low-molecular-weight phosphatase substrate para-nitrophenylphosphate, and its activity was inhibited by sodium vanadate. More importantly, our study also revealed that both FYVE-DSP1 and FYVE-DSP2 efficiently and specifically dephosphorylated phosphotidylinositol 3-phosphate. Subcellular fractionation demonstrated partition of FYVE-DSP1 and FYVE-DSP2 in membrane fractions, and immunofluorescent cell staining showed perinuclear localization of the enzymes. FYVE-DSP2 is expressed in many human tissues with an alternatively spliced isoform expressed in the kidney. Together with two homologous hypothetical proteins found in Caenorhabditis elegans and Drosophila, FYVE-DSP1 and FYVE-DSP2 form a subfamilyof phosphatases that may have an importantrole in cellular processes.

Adenoviridae↗

PubMed2001

Retraction.

Explore the source record for details and available documents.

Retraction Notice↗

PubMed2001

Molecular cloning and characterization of SPAP1, an inhibitory receptor.

We have cloned a novel cell-surface protein designated SPAP1a for SH2 domain-containing phosphatase anchor protein 1a. SPAP1a belongs to the group of type I transmembrane proteins. Its extracellular domain contains a single immunoglobulin-like domain, and its intracellular segment has two immunoreceptor tyrosine-based inhibition motifs (ITIMs). We also identified two alternatively spliced products that were named SPAP1b and SPAP1c. SPAP1b contains a short intracellular part without ITIMs, while SPAP1c lacks the transmembrane segment and represents a potential soluble protein. Sequence alignment with the genomic database revealed that the SPAP1 gene contains seven exons and is localized at chromosome 1q21. PCR analyses demonstrated that SPAP1a mRNA is specifically expressed in human hematopoietic tissues including spleen, peripheral blood, and bone marrow, and it may be restricted to expression in B cells. Recombinant SPAP1a is tyrosine phosphorylated in cells upon pervanadate stimulation and tyrosine-phosphorylated SPAP1a recruits the SH2 domain containing phosphatase SHP-1, but not SHP-2. As a specific anchor protein of SHP-1, SPAP1a may have an important role in hematopoietic cell signaling.

Alternative Splicing↗

PubMed2001

Cell density regulates tyrosine phosphorylation and localization of focal adhesion kinase.

We have investigated tyrosine phosphorylation of cellular proteins at different cell densities. A tyrosine-phosphorylated protein of 120 kDa was detected when cells were plated sparsely. The phosphorylation level of the protein gradually declined as the cells were plated at higher densities or when the sparsely plated cells approached confluence. This density-dependent phosphorylation was also associated with cell attachment since it disappeared when the cells were detached from plates or when the cells were cultured in suspension. Immunoblotting and immunoprecipitation analyses with specific antibodies revealed that the 120-kDa protein corresponded to the focal adhesion kinase (FAK) and the protein level of FAK was not altered at different cell densities. In vitro kinase assays demonstrated that the kinase activity of FAK decreased with increasing cell densities in parallel with its dephosphorylation. Cell density also affects localization of FAK associated with rearrangement of actin stress fibers. At low cell densities, FAK and actin stress fiber are distributed around the periphery of cells while they are dispersed over the ventral surface in high-density cells. Finally, the density-regulated tyrosine phosphorylation and localization of FAK appeared to be mediated by an insoluble factor produced by high-density cells.

3T3 Cells↗

PubMed2001

Relation between the flexibility of the WPD loop and the activity of the catalytic domain of protein tyrosine phosphatase SHP-1.

The conserved WPD loop of protein tyrosine phosphatases play an important role in the catalytic activity and the invariant aspartate residue acts as a general acid/base catalyst in the dephosphorylation reaction. In our previous report, we have demonstrated that the catalytic activities of the PTPs are influenced by the flexibility and stability of the WPD loop in its active "open" conformation [Yang et al., 1998]. Phosphatases with a more flexible WPD loop generally have higher specific activity. In this report, we modify the WPD loop of SHP-1 by alanine-scan mutation of the residues flanking the loop and measure their effects on the catalytic activity of the phosphatase. We show that the S418A, V424A, S426A, E427A, and P428A mutants increase the phosphatase activity, possibly due to the increased flexibility of the WPD loop, whereas the L417A, L417G and P425A mutants decrease its phosphatase activity. In addition, we propose that the two-proline residues in the WPD loop (Pro(420) and Pro(425) in SHP-1) work as pivotal points through a conserved hydrophobic network and allows residues between the pivotal points to have maximum flexibility in enhancing the phosphatase activity. Furthermore, our data suggest that the hydrolysis of the phosphoryl-cysteine intermediate, not its formation, is the rate-limiting step with p-nitrophenyl phosphate as the substrate while both the steps are rate-limiting with phosphotyrosine as the substrate.

Alanine↗

PubMed2000

The protein-tyrosine phosphatase SHP-2 is required during angiotensin II-mediated activation of cyclin D1 promoter in CHO-AT1A cells.

Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary (CHO) cells stably expressing a rat vascular angiotensin II type 1A receptor (CHO-AT(1A)). Cyclin D1 protein expression is regulated by mitogens, and its assembly with the cyclin-dependent kinases induces phosphorylation of the retinoblastoma protein pRb, a critical step in G(1) to S phase cell cycle progression contributing to the proliferative responses. In the present study, we found that in CHO-AT(1A) cells, Ang II induced a rapid and reversible tyrosine phosphorylation of various intracellular proteins including the protein-tyrosine phosphatase SHP-2. Ang II also induced cyclin D1 protein expression in a phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner. Using a pharmacological and a co-transfection approach, we found that p21(ras), Raf-1, phosphatidylinositol 3-kinase and also the catalytic activity of SHP-2 and its Src homology 2 domains are required for cyclin D1 promoter/reporter gene activation by Ang II through the regulation of MAPK/ERK activity. Our findings suggest for the first time that SHP-2 could play an important role in the regulation of a gene involved in the control of cell cycle progression resulting from stimulation of a G protein-coupled receptor independently of epidermal growth factor receptor transactivation.

Angiotensin II↗

PubMed2000

Identification and characterization of leukocyte-associated Ig-like receptor-1 as a major anchor protein of tyrosine phosphatase SHP-1 in hematopoietic cells.

SHP-1, an SH2 domain-containing tyrosine phosphatase, has a crucial role in hematopoiesis. Here we report that SHP-1 is associated with two major tyrosine-phosphorylated proteins in hematopoietic cells treated with the tyrosine phosphatase inhibitor, pervanadate. One of the proteins corresponds to leukocyte-associated Ig-like receptor-1 (LAIR-1), a recently cloned transmembrane protein. Molecular cloning revealed four isoforms of the protein. LAIR-1 is hyper-phosphorylated on tyrosyl residues in cells overexpressing a catalytically inactive mutant form of SHP-1 as well as in pervanadate-treated cells. An antibody against the extracellular domain of the protein also induced its tyrosine phosphorylation. Tyrosine-phosphorylated LAIR-1 specifically interacts with SHP-1 but not with SHP-2, a structurally related tyrosine phosphatase. Using site-specific mutagenesis, we demonstrated that Tyr(233) and Tyr(263), each embedded in an immunoreceptor tyrosine-based inhibitory motif, are responsible for tyrosine phosphorylation of LAIR-1 and recruitment of SHP-1. Both tyrosyl residues are required for SHP-1 binding. Protein kinases responsible for tyrosine phosphorylation of LAIR-1 may belong to the Src family since PP1, a Src family kinase inhibitor, significantly inhibited its phosphorylation. As a major binding protein of SHP-1 on the plasma membrane, LAIR-1 may play an important role in hematopoietic cell signaling.

Amino Acid Sequence↗

PubMed2000

FYVE-DSP1, a dual-specificity protein phosphatase containing an FYVE domain.

Dual-specificity protein phosphatases (DSPs) dephosphorylate proteins at Ser/Thr and Tyr. FYVE domain is a double zinc finger motif which specifically binds phosphatidylinositol(3)-phosphate. Here, we report a novel dual specificity phosphatase that contains a FYVE domain at the C-terminus. We designate the protein FYVE-DSP1. Molecular cloning yielded three isoforms of the enzyme presumably derived from alternate RNA splicing. Sequence alignment revealed that the catalytic phosphatase domain of FYVE-DSP1 closely resembled that of myotubularin, while its FYVE domain has all the conserved amino acid residues found in other proteins of the same family. Recombinant FYVE-DSP1 is partitioned in both cytosolic and membrane fractions. It dephosphorylates proteins phosphorylated on Ser, Thr, and Tyr residues and low molecular weight phosphatase substrate para-nitrophenylphosphate. It shows typical characteristics of other DSPs and protein tyrosine phosphatases (PTPs). These include inhibition by sodium vanadate and pervanadate, pH dependency, and inactivation by mutation of the key cysteinyl residue at the phosphatase signature motif. Finally, PCR analyses demonstrated that FYVE-DSP1 is widely distributed in human tissues but different spliced forms expressed differently.

Alternative Splicing↗

PubMed2000

Regulation of calcium-sensitive tyrosine kinase Pyk2 by angiotensin II in endothelial cells. Roles of Yes tyrosine kinase and tyrosine phosphatase SHP-2.

Calcium-sensitive tyrosine kinase Pyk2 has been implicated in the regulation of ion channels, cellular adhesion, and mitogenic and hypertrophic reactions. In this study, we have investigated the regulation of Pyk2 by angiotensin II (Ang II) in pulmonary vein endothelial cells. We found that the Ang II-induced tyrosine phosphorylation of Pyk2, which requires the activity of Src family kinase, was specifically regulated by the Src family kinase member, Yes kinase. Moreover, we identified for the first time the constitutive association of Pyk2 with an Src homology 2 (SH2) domain-containing tyrosine phosphatase SHP-2. SHP-2 interacts with Pyk2 through a region other than its SH2 domains. Pyk2 can be dephosphorylated in vitro in SHP-2 immunoprecipitates and in intact cells expressing an NH(2) terminus-truncated form of SHP-2, which lacks the two SH2 domains but has an enhanced phosphatase activity. Ang II activates the endogenous SHP-2. Finally, the SHP-2-mediated dephosphorylation of Pyk2 correlates with the negative effect of SHP-2 on the Ang II-induced activation of extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase. Thus, the balance of Pyk2 tyrosine phosphorylation in response to Ang II is controlled by Yes kinase and by a tyrosine phosphatase SHP-2 in endothelial cells.

Angiotensin II↗

PubMed2000

Dissecting the interaction of SHP-2 with PZR, an immunoglobulin family protein containing immunoreceptor tyrosine-based inhibitory motifs.

Tyrosine phosphorylation of membrane proteins plays a crucial role in cell signaling by recruiting Src homology 2 (SH2) domain-containing signaling molecules. Recently, we have isolated a transmembrane protein designated PZR that specifically binds tyrosine phosphatase SHP-2, which has two SH2 domains (Zhao, Z. J., and Zhao, R. (1998) J. Biol. Chem. 273, 29367-29372). PZR belongs to the immunoglobulin superfamily. Its intracellular segment contains four putative sites of tyrosine phosphorylation. By site-specific mutagenesis, we found that the tyrosine 241 and 263 embedded in the consensus immunoreceptor tyrosine-based inhibitory motifs VIYAQL and VVYADI, respectively, accounted for the entire tyrosine phosphorylation of PZR. The interaction between PZR and SHP-2 requires involvement of both tyrosyl residues of the former and both SH2 domains of the latter, since its was disrupted by mutating a single tyrosyl residue or an SH2 domain. Overexpression of catalytically inactive but not active forms of SHP-2 bearing intact SH2 domains in cells caused hyperphosphorylation of PZR. In vitro, tyrosine-phosphorylated PZR was efficiently dephosphorylated by the full-length form of SHP-2 but not by its SH2 domain-truncated form. Together, the data indicate that PZR serves not only as a specific anchor protein of SHP-2 on the plasma membrane but also as a physiological substrate of the enzyme. The coexisting binding and dephosphorylation of PZR by SHP-2 may function to terminate signal transduction initiated by PZR and SHP-2 and to set a threshold for the signal transduction to be initiated.

Blotting, Western↗

PubMed2000

Structural basis for substrate specificity of protein-tyrosine phosphatase SHP-1.

The substrate specificity of the catalytic domain of SHP-1, an important regulator in the proliferation and development of hematopoietic cells, is critical for understanding the physiological functions of SHP-1. Here we report the crystal structures of the catalytic domain of SHP-1 complexed with two peptide substrates derived from SIRPalpha, a member of the signal-regulatory proteins. We show that the variable beta5-loop-beta6 motif confers SHP-1 substrate specificity at the P-4 and further N-terminal subpockets. We also observe a novel residue shift at P-2, the highly conserved subpocket in protein- tyrosine phosphatases. Our observations provide new insight into the substrate specificity of SHP-1.

Animals↗

PubMed2000

Tyrosine phosphorylation of myelin P(0) and its implication in signal transduction.

P(0), a major structural protein of peripheral myelin, belongs to the immunoglobulin superfamily. Sequence comparison of P(0) with PZR, a tyrosine phosphatase SHP-2 binding protein we recently cloned, revealed the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular portion of the P(0) molecule. To study the role of this putative ITIM in signal transduction, we have expressed P(0) in HT-1080 and 293 cells. Stimulation of the transfected cells with pervanadate, a powerful inhibitor of tyrosine phosphatases, resulted in tyrosine phosphorylation of P(0) and its association with several tyrosine-phosphorylated proteins. Mutation of Y(220) embedded in the ITIM to phenylalanine abolished the tyrosine phosphorylation and the association. Tyrosine phosphorylation of P(0) and its association with other signaling proteins were also observed in pervanadate-treated RN22 Schwannoma cells, which express endogenous P(0). Furthermore, injection of pervanadate induced tyrosine phosphorylation of P(0) in peripheral nerves of newborn but not adult mice. The physiological importance of the ITIM in P(0) is implied by the fact that a naturally occurred P(0) mutant with a disrupted ITIM has a dominant role in causing Dejerine-Scotts syndrome. Taken together, P(0) is phosphorylated on Try(220). The presence of an ITIM in P(0) and its ability to mediate protein-protein interaction through tyrosine phosphorylation indicate that P(0) is not merely a structural protein but may also be a crucial player in cell signaling.

Amino Acid Sequence↗

PubMed2000

Stem cell factor and erythropoietin inhibit apoptosis of human erythroid progenitor cells through different signalling pathways.

Erythropoietin (EPO) and stem cell factor (SCF) are two important factors in human erythropoiesis. We have recently demonstrated that SCF and EPO synergistically activate mitogen-activated protein (MAP) kinase, thereby promoting growth of human erythroid colony-forming cells (ECFCs). In the present study, we have examined the intracellular mechanisms by which SCF and EPO maintain survival of these cells. In the absence of SCF and EPO, human ECFCs underwent rapid apoptosis. The process was significantly inhibited by addition of a single factor and was totally prevented in the presence of both factors. Treatment of ECFCs with wortmannin, a specific inhibitor of phosphoinositide 3-kinase (PI3K), inhibited the antiapoptotic effect of SCF but had no effect on that of EPO, indicating that SCF but not EPO inhibits apoptosis through the PI3K pathway. In contrast, treatment of ECFCs with PD98059, a specific inhibitor of MAP kinase/ERK kinase (MEK), inhibited cell growth but had no effect on the antiapoptotic activity of either SCF or EPO, suggesting that SCF and EPO prevent apoptosis of human ECFCs independent of the extracellular signal-regulated kinase (ERK) pathway. Interestingly, both EPO and SCF induced activation of PI3K. However, through PI3K, SCF caused activation of protein kinase B (PKB), an anti-apoptosis signal, whereas EPO led to activation of ERKs. Furthermore, the SCF- and EPO-maintained expression of antiapoptotic protein Bcl-XL was correlated with the activation of ERKs and was inhibited by PD98059, suggesting that Bcl-XL may not have a major role in preventing apoptosis of human ECFCs. Phosphorylated BAD was not affected by SCF, EPO or wortmannin. Taken together with our previous results, the present study indicates that SCF and EPO support survival and growth of human ECFCs through different signalling pathways and that they transduce distinctly different signals through activation of PI3K.

Androstadienes↗

PubMed1999

Distinct roles of JNKs/p38 MAP kinase and ERKs in apoptosis and survival of HCD-57 cells induced by withdrawal or addition of erythropoietin.

Erythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.

Animals↗

PubMed1999

Fyn kinase-directed activation of SH2 domain-containing protein-tyrosine phosphatase SHP-2 by Gi protein-coupled receptors in Madin-Darby canine kidney cells.

SHP-2, an SH2 domain-containing protein-tyrosine phosphatase, plays an important role in receptor tyrosine kinase-regulated cell proliferation and differentiation. Little is known about the activation mechanisms and the participation of SHP-2 in the activity of G protein-coupled receptors lacking intrinsic tyrosine kinase activity. We show that the activity of SHP-2 (but not SHP-1) is specifically stimulated by the selective alpha2A-adrenergic receptor agonist UK14304 and by lysophosphatidic acid (LPA) in Madin-Darby canine kidney (MDCK) cells. UK14304 and LPA promote the tyrosine phosphorylation of SHP-2 and its association with Grb2. The agonist-induced direct interaction of Grb2 with SHP-2 is mediated by the SH2 domain of Grb2 and the tyrosine phosphorylation of SHP-2. Rapid activation of Src family kinase by UK14304 preceded the SHP-2 activation. Among the Src family members (Src, Fyn, Lck, Yes, and Lyn) present in MDCK cells, Fyn was the only one specifically associated with SHP-2, and the physical interaction between them, which requires the Src family kinase activity, was increased in response to the agonists. Pertussis toxin, PP1 (a selective Src family kinase inhibitor), or overexpression of a catalytically inactive mutant of Fyn blocked the UK14304- or LPA-stimulated activity of SHP-2, SHP-2 tyrosine phosphorylation, and SHP-2 association with Grb2. Therefore, we have demonstrated for the first time that the activation of SHP-2 by these Gi protein-coupled receptors requires Fyn kinase and that there is a specific physical interaction of Fyn kinase with SHP-2 in MDCK cells.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Tyrosine phosphatase SHP-2 dephosphorylates the platelet-derived growth factor receptor but enhances its downstream signalling.

SHP-2 is a widely distributed Src homology 2 (SH2) domain-containing tyrosine phosphatase that is recruited to growth factor receptors on stimulation. We have transiently co-expressed several catalytically active and inactive forms of the enzyme with the platelet-derived growth factor (PDGF) receptor in human embryonic kidney 293 cells. The catalytically active forms of SHP-2 decreased the tyrosine phosphorylation of the receptor, whereas the catalytically inactive forms increased the phosphorylation. However, PDGF-induced activation of the mitogen-activated protein (MAP) kinase pathway was enhanced by the active forms of SHP-2 but decreased by the inactive forms. The results suggest that the PDGF receptor is a physiological substrate of SHP-2 and that SHP-2 has a positive role in the PDGF-stimulated activation of MAP kinase. The dissociation of the receptor phosphorylation from the activation of MAP kinase suggests that signalling through growth factor receptors does not depend merely on their tyrosine phosphorylation.

Calcium-Calmodulin-Dependent Protein Kinases↗