4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3- yl) vinyl)-2-methoxy-phenol) (CNB-001) Does Not Regulate Human Recombinant Protein-Tyrosine Phosphatase1B (PTP1B) Activity in vitro

ABSTRACT Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains one such CpsB-like PTP, YwqE, in addition to two class II Cys-based PTPs, YwlE and YfkJ. The substrates for both YwlE and YfkJ are presently unknown, while YwqE was shown to dephosphorylate two phosphotyrosine-containing proteins implicated in UDP-glucuronate biosynthesis, YwqD and YwqF. In this study, we characterize YwqE, compare the activities of the three B. subtilis PTPs (YwqE, YwlE, and YfkJ), and demonstrate that the two B. subtilis class II PTPs do not dephosphorylate the physiological substrates of YwqE.

Download Full-text

Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains.

The Journal of Cell Biology ◽

10.1083/jcb.130.5.1181 ◽

1995 ◽

Vol 130 (5) ◽

pp. 1181-1187 ◽

Cited By ~ 404

Author(s):

M D Schaller ◽

C A Otey ◽

J D Hildebrand ◽

J T Parsons

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Cytoplasmic Domain ◽

Embryo Cell ◽

Vitro Assay ◽

Protein Tyrosine ◽

Signaling Complex

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.

Download Full-text

Protein-tyrosine phosphatase activity of CD45 is activated by sequential phosphorylation by two kinases

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5523-5532.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5523-5532

Author(s):

D R Stover ◽

K A Walsh

Keyword(s):

T Cell Activation ◽

Protein Tyrosine Phosphatase ◽

Time Course ◽

Cell Activation ◽

Regulatory Mechanism ◽

Transmembrane Protein ◽

Tyrosine Phosphatase ◽

Protein Tyrosine

We describe a potential regulatory mechanism for the transmembrane protein-tyrosine phosphatase CD45. Phosphorylation on both tyrosine and serine residues in vitro results in an activation of CD45 specifically toward one artificial substrate but not another. The activation of these kinases appears to be order dependent, as it is enhanced when phosphorylation of tyrosine precedes that of serine but phosphorylation in the reverse order yields no activation. Any of four protein-tyrosine kinases tested, in combination with the protein-serine/threonine kinase, casein kinase II, was capable of mediating this activation in vitro. The time course of phosphorylation of CD45 in response to T-cell activation is consistent with the possibility that this regulatory mechanism is utilized in vivo.

Download Full-text

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.147-155.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 147-155

Author(s):

B S Cobb ◽

M D Schaller ◽

T H Leu ◽

J T Parsons

Keyword(s):

Focal Adhesion ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Src Family Kinases ◽

Protein Tyrosine Kinases ◽

Protein Tyrosine ◽

Sh2 Domains ◽

Embryo Cells ◽

Stable Association

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.

Download Full-text

Differential Regulation of the Cell Wall Integrity Mitogen-Activated Protein Kinase Pathway in Budding Yeast by the Protein Tyrosine Phosphatases Ptp2 and Ptp3

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7651 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7651-7660 ◽

Cited By ~ 72

Author(s):

Christopher P. Mattison ◽

Scott S. Spencer ◽

Kurt A. Kresge ◽

Ji Lee ◽

Irene M. Ota

Keyword(s):

Cell Wall ◽

Protein Tyrosine Phosphatases ◽

Negative Regulator ◽

Cell Wall Integrity ◽

Dependent Manner ◽

Tyrosine Phosphatases ◽

Protein Tyrosine ◽

Growth Defects ◽

Mitogen Activated Protein

ABSTRACT Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity and protein tyrosine phosphatases (PTPs) in yeasts. InSaccharomyces cerevisiae, two PTPs, Ptp2 and Ptp3, inactivate the MAPKs, Hog1 and Fus3, with different specificities. To further examine the functions and substrate specificities of Ptp2 and Ptp3, we tested whether they could inactivate a third MAPK, Mpk1, in the cell wall integrity pathway. In vivo and in vitro evidence indicates that both PTPs inactivate Mpk1, but Ptp2 is the more effective negative regulator. Multicopy expression of PTP2, but not PTP3, suppressed growth defects due to the MEK kinase mutation, BCK1-20, and the MEK mutation,MKK1-386, that hyperactivate this pathway. In addition, deletion of PTP2, but not PTP3, exacerbated growth defects due to MKK1-386. Other evidence supported a role for Ptp3 in this pathway. Expression of MKK1-386 was lethal in the ptp2Δ ptp3Δ strain but not in either single PTP deletion strain. In addition, the ptp2Δ ptp3Δ strain showed higher levels of heat stress-induced Mpk1-phosphotyrosine than the wild-type strain or strains lacking either PTP. The PTPs also showed differences in vitro. Ptp2 was more efficient than Ptp3 at binding and dephosphorylating Mpk1. Another factor that may contribute to the greater effectiveness of Ptp2 is its subcellular localization. Ptp2 is predominantly nuclear whereas Ptp3 is cytoplasmic, suggesting that active Mpk1 is present in the nucleus. Last, PTP2 but not PTP3 transcript increased in response to heat shock in a Mpk1-dependent manner, suggesting that Ptp2 acts in a negative feedback loop to inactivate Mpk1.

Download Full-text

Both cell-surface carbohydrates and protein tyrosine phosphatase are involved in the differentiation of astrocytes in vitro

Glia ◽

10.1002/1098-1136(200010)32:1<60::aid-glia60>3.0.co;2-5 ◽

2000 ◽

Vol 32 (1) ◽

pp. 60-70 ◽

Cited By ~ 11

Author(s):

Tasuku Sasaki ◽

Tamao Endo

Keyword(s):

Cell Surface ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Protein Tyrosine ◽

Surface Carbohydrates

Download Full-text

Novaferon, a novel recombinant protein produced by DNA-shuffling of IFN-α, shows antitumor effect in vitro and in vivo

Cancer Cell International ◽

10.1186/1475-2867-14-8 ◽

2014 ◽

Vol 14 (1) ◽

pp. 8 ◽

Cited By ~ 6

Author(s):

Meng Li ◽

Chunming Rao ◽

Dening Pei ◽

Lan Wang ◽

Yonghong Li ◽

...

Keyword(s):

Recombinant Protein ◽

Antitumor Effect ◽

Dna Shuffling

Download Full-text

Signal transduction mechanisms involved in in vitro ram sperm capacitation

Reproduction ◽

10.1530/rep.1.00770 ◽

2006 ◽

Vol 132 (5) ◽

pp. 721-732 ◽

Cited By ~ 45

Author(s):

Patricia Grasa ◽

José Álvaro Cebrián-Pérez ◽

Teresa Muiño-Blanco

Keyword(s):

Tyrosine Phosphorylation ◽

Calcium Influx ◽

Calcium Entry ◽

Calcium Entry Blocker ◽

Protein Tyrosine Phosphorylation ◽

Sperm Capacitation ◽

Protein Tyrosine ◽

Entry Blocker ◽

Ram Sperm

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

Download Full-text