Genetic Encoding of A Nonhydrolyzable Phosphotyrosine Analog in Mammalian Cells

Protein tyrosine phosphorylation plays a critical role in signal transduction and the regulation of many cellular processes. It is of great significance to understand the underlying regulatory mechanism of particular tyrosine phosphorylation events. Here we report the genetic incorporation of a phosphotyrosine (pTyr) analog, p-carboxymethyl-L-phenylalanine (CMF), into proteins in mammalian cells. This nonhydrolyzable pTyr analog can facilitate biological studies by removing complications caused by the dynamic interconversion between the phosphorylated and non-phosphorylated isoforms of a protein. The developed methodology was demonstrated by using the human signal transducer and activator of transcription-1 (STAT1) as a model protein for homogeneous and defined incorporation of CMF. This tool will greatly enhance our capability to study protein tyrosine phosphorylation-associated biomolecular and cellular events, and enhance biomedical research that target protein tyrosine phosphorylation, which will have a broad impact to both fundamental studies and practical applications.

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IL-5 receptor-mediated tyrosine phosphorylation of SH2/SH3-containing proteins and activation of Bruton's tyrosine and Janus 2 kinases.

Journal of Experimental Medicine ◽

10.1084/jem.180.6.2101 ◽

1994 ◽

Vol 180 (6) ◽

pp. 2101-2111 ◽

Cited By ~ 118

Author(s):

S Sato ◽

T Katagiri ◽

S Takaki ◽

Y Kikuchi ◽

Y Hitoshi ◽

...

Keyword(s):

Signal Transduction ◽

B Cell ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Catalytic Domain ◽

Critical Role ◽

Phosphatidyl Inositol ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Interleukin 5

Interleukin 5 (IL-5) induces proliferation and differentiation of B cells and eosinophils by interacting with its receptor (IL-5R) which consists of two distinct polypeptide chains, alpha and beta (beta c). Although both IL-5R alpha and beta c lack a kinase catalytic domain, IL-5 is capable of inducing tyrosine phosphorylation of cellular proteins. We investigated the role of IL-5R alpha in tyrosine phosphorylation of molecules involved in IL-5 signal transduction, using an IL-5-dependent early B cell line, Y16 and transfectants expressing intact or mutant IL-5R alpha together with intact beta c. The results revealed that the transfectants expressing truncated IL-5R alpha, which entirely lacks a cytoplasmic domain, together with beta c, showed neither protein-tyrosine phosphorylation nor proliferation in response to IL-5. This confirms that IL-5R alpha plays a critical role in protein-tyrosine phosphorylation which triggers cell growth. IL-5 stimulation results in rapid tyrosine phosphorylation of beta c and proteins containing Src homology 2 (SH2) and/or SH3 domains such as phosphatidyl-inositol-3 kinase, Shc, Vav, and HS1, suggesting their involvement in IL-5-mediated signal transduction. IL-5 stimulation significantly enhanced activities of Janus 2 and B cell-specific Bruton's tyrosine kinases (JAK2 and Btk) and increased the tyrosine phosphorylation of JAK2 kinase. These results and recent data on signaling of growth factors taken together, multiple biochemical pathways driven by tyrosine kinases such as JAK2 and Btk are involved in IL-5 signal transduction.

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Recent advances in PTP1B signaling in metabolism and cancer

Bioscience Reports ◽

10.1042/bsr20211994 ◽

2021 ◽

Author(s):

Olga Villamar-Cruz ◽

Marco A. Loza-Mejía ◽

Luis E. Arias-Romero ◽

Ignacio Camacho-Arroyo

Keyword(s):

Signaling Pathways ◽

Tyrosine Phosphorylation ◽

Cancer Progression ◽

Tyrosine Kinases ◽

Critical Role ◽

Protein Tyrosine Phosphatases ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Promising Therapeutic Target ◽

Molecular Aspects

Protein tyrosine phosphorylation is one of the major post-translational modifications in eukaryotic cells and represents a critical regulatory mechanism of a wide variety of signaling pathways. Aberrant protein tyrosine phosphorylation has been linked to various diseases, including metabolic disorders and cancer. Few years ago, protein tyrosine phosphatases (PTPs) were considered as tumor suppressors, able to block the signals emanated from receptor tyrosine kinases. However, recent evidence demonstrates that a misregulation of PTPs activity plays a critical role in cancer development and progression. Here, we will focus on PTP1B, an enzyme that has been linked to the development of type 2 diabetes and obesity through the regulation of insulin and leptin signaling, and with a promoting role in the development of different types of cancer through the activation of several pro-survival signaling pathways. In this review, we discuss the molecular aspects that support the crucial role of PTP1B in different cellular processes underlying diabetes, obesity and cancer progression, and its visualization as a promising therapeutic target.

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Antibody Array Platform to Monitor Protein Tyrosine Phosphorylation in Mammalian Cells

Phospho-Proteomics - Methods in Molecular Biology ◽

10.1007/978-1-60327-834-8_18 ◽

2009 ◽

pp. 247-255 ◽

Cited By ~ 7

Author(s):

Alicia S. Chung ◽

Y. Eugene Chin

Keyword(s):

Tyrosine Phosphorylation ◽

Mammalian Cells ◽

Antibody Array ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Array Platform

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Protein-tyrosine Kinase p72 syk Is Activated by Platelet Activating Factor in Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648987 ◽

1994 ◽

Vol 72 (06) ◽

pp. 937-941 ◽

Cited By ~ 7

Author(s):

Karim Rezaul ◽

Shigeru Yanagi ◽

Kiyonao Sada ◽

Takanobu Taniguchi ◽

Hirohei Yamamura

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Inhibitor ◽

Critical Role ◽

Platelet Activating Factor ◽

Kinase Assay ◽

Potential Candidate ◽

Protein Tyrosine ◽

Induced Aggregation

SummaryIt has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p72 syk is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72 syk which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72 syk coincided with activation of yllsyk. Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72 syk .Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72 syk activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72 syk may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.

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