Purification and characterization of a human recombinant T-cell protein-tyrosine-phosphatase from a baculovirus expression system

Zander, Norbert; Lorenzen, James A.; Cool, D. E.; Tonks, Nicholas K.; Daum, Günter; Krebs, Edwin G.; Fischer, Edmond H.

doi:10.1021/bi00242a022

Cited by 71 publications

(47 citation statements)

References 37 publications

(39 reference statements)

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“…In the above-mentioned publication it was also observed that the activity of the T-cell PTPase against phosphorylated RCML is suppressed by the C-terminus (Zander et al, 1991). Although the differences are modest, our data suggest that the C-terminus of PTPlB also exerts an inhibitory influence on the activity.…”

Section: Discussionsupporting

confidence: 62%

Expression, purification and crystallization of human phosphotyrosine phosphatase 1B

Hoppe

Berne

Stock

et al. 1994

European Journal of Biochemistry

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Protein phosphotyrosine phosphatases are believed to be involved in the regulation of the activity of cellular proteins, such as receptor tyrosine kinases, by controlling their phosphorylation status. One of the best described and characterized protein of this class of enzymes is the phosphotyrosine phosphatase 1B. To obtain sufficient quantities for structural investigations, truncated forms of PTPlB encompassing the catalytic domain were over-expressed in Escherichia coli and purified to apparent homogeneity by conventional chromatography. The activity of these purified enzymes has been compared with the wild-type enzyme expressed in mammalian cells. By measuring the activities against p-nitrophenyl phosphate, the pH dependence of this activity, and responses to different modulators, it could be demonstrated that the truncated forms of PTPlB retained the same characteristics as the full-length mammalian enzyme, but are not subject to inhibition of enzymic activity mediated by the C-terminus. Due to their improved solubility, it can be assumed that the catalytic domains are advantageous for crystallization studies in comparison to the natural enzyme. In a screening for crystallization conditions, we obtained protein crystals indicating that the quality of the purified protein is sufficient for crystallographic studies.

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Section: Discussionsupporting

confidence: 62%

Expression, purification and crystallization of human phosphotyrosine phosphatase 1B

Hoppe

Berne

Stock

et al. 1994

European Journal of Biochemistry

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“…8A). PTP1B and TCPTP, which display comparable specific activities in assays in vitro (55,62), were overexpressed in the p210 bcr-abl transfectants (Fig. 8C and D).…”

Section: Resultsmentioning

confidence: 90%

Protein Tyrosine Phosphatase 1B Antagonizes Signalling by Oncoprotein Tyrosine Kinase p210 bcr-abl In Vivo

LaMontagne

Flint

Franza

et al. 1998

Molecular and Cellular Biology

102

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The p210 bcr-abl protein tyrosine kinase (PTK) appears to be directly responsible for the initial manifestations of chronic myelogenous leukemia (CML). In contrast to the extensive characterization of the PTK and its effects on cell function, relatively little is known about the nature of the protein tyrosine phosphatases (PTPs) that may modulate p210 bcr-abl-induced signalling. In this study, we have demonstrated that expression of PTP1B is enhanced specifically in various cells expressing p210 bcr-abl, including a cell line derived from a patient with CML. This effect on expression of PTP1B required the kinase activity of p210 bcr-abl and occurred rapidly, concomitant with maximal activation of a temperature-sensitive mutant of the PTK. The effect is apparently specific for PTP1B since, among several PTPs tested, we detected no change in the levels of TCPTP, the closest relative of PTP1B. We have developed a strategy for identification of physiological substrates of individual PTPs which utilizes substrate-trapping mutant forms of the enzymes that retain the ability to bind to substrate but fail to catalyze efficient dephosphorylation. We have observed association between a substrate-trapping mutant of PTP1B (PTP1B-D181A) and p210 bcr-abl, but not v-Abl, in a cellular context. Consistent with the trapping data, we observed dephosphorylation of p210 bcr-abl, but not v-Abl, by PTP1B in vivo. We have demonstrated that PTP1B inhibited binding of the adapter protein Grb2 to p210 bcr-abl and suppressed p210 bcr-abl-induced transcriptional activation that is dependent on Ras. These results illustrate selectivity in the effects of PTPs in a cellular context and suggest that PTP1B may function as a specific, negative regulator of p210 bcr-abl signalling in vivo.

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“…The nature of the subcellular localization of the T-cell PTPb has yet to be clarified. However, both rat PTPase 1 and the human placental PTPase 1B are targeted to the endoplasmic reticulum (10,37 (7), since no basic cluster separated by the consensus 10-amino-acid spacer region exists in the carboxyl-terminal region between amino acids 345 and 369. The results of these experiments indicate that the MPTP NLS resembles the NLS motifs present in simian virus 40 large T antigen (7,29) and in the nucleolus-localized human immunodeficiency virus Tat protein (30).…”

Section: Discussionmentioning

confidence: 99%

“…Although isolated from a human T-cell cDNA library, this enzyme is ubiquitously expressed in human tissues. In vitro expression of the cDNA yields an inactive enzyme that can be activated by trypsin cleavage of a hydrophobic carboxyl terminus (37). This stretch of hydrophobic amino acids is thought to anchor the enzyme to high-molecular-weight complexes from which it can be released and activated by proteolytic processing.…”

mentioning

confidence: 99%

Nuclear localization and cell cycle regulation of a murine protein tyrosine phosphatase.

Tillmann¹,

Wagner²,

Boerboom³

et al. 1994

Mol. Cell. Biol.

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MPTP is a murine homolog of the human T-cell protein tyrosine phosphatase (PTPase) In the past decade, an enormous amount of information has been gathered on the multifaceted involvement of protein phosphorylation in biology. Tyrosine phosphorylation has drawn special attention because of its role in key processes such as signal transduction, cellular differentiation, and cell cycle progression. Through these processes, it has been shown to influence growth, development, and oncogenic transformation. The level of tyrosine phosphorylation results from the activity of both tyrosine-specific kinases and phosphatases. The importance of protein tyrosine phosphatases (PTPases) has been recognized more recently by the cloning and characterization of an ever-growing number of these genes. Structurally, the PTPase family can be subdivided into two groups: receptorlike PTPases and intracellular enzymes (9). With some exceptions, the receptor-like enzymes feature large and diverse extracellular domains and two tandemly arranged intracellular PTPase conserved domains. Much like their receptor phosphotyrosine kinase counterparts, most bind as yet undefined ligands at their extracellular domains. The cloning of intracellular PTPases that possess SH2 domains provides a clue about the molecular function of at least a small subset of these polypeptides (27). For example, the SH2-containing PTPase corkscrew is involved in a signal transduction pathway responsible for cell fate at the termini of Drosophila embryos (22).

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Purification and characterization of a human recombinant T-cell protein-tyrosine-phosphatase from a baculovirus expression system

Cited by 71 publications

References 37 publications

Expression, purification and crystallization of human phosphotyrosine phosphatase 1B

Expression, purification and crystallization of human phosphotyrosine phosphatase 1B

Protein Tyrosine Phosphatase 1B Antagonizes Signalling by Oncoprotein Tyrosine Kinase p210 bcr-abl In Vivo

Nuclear localization and cell cycle regulation of a murine protein tyrosine phosphatase.

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