Biochemical and Biophysical Research Communications
Identification and characterization of two novel low-molecular-weight dual specificity phosphatases☆
Section snippets
Materials and methods
Materials. HA-ERK2, HA-JNK1, and HA-p38α were gifts from Roger Davis. pET21a/MKP3-His6 and pET15b-CL100(MKP1) were gifts from Zhong-Yin Zhang. Human recombinant epidermal growth factor was obtained from Upstate Biotechnology (01-107). Anisomycin was obtained from Sigma (A9789). p44/p42 MAP kinase (9800), SAPK/JNK (9810), and p38 MAP kinase (9820) non-radioactive IP-kinase assay kits and inclusive antibodies were obtained from Cell Signaling Technology. Synthetic MAPK phosphopeptides were
Identification of two novel dual specificity phosphatases
Although they comprise a diverse group of proteins, PTPs can be easily recognized based on the strong sequence conservation within their catalytic domains. We therefore expected that many expressed sequence tags (ESTs) representing novel PTPs would contain precomputed annotation based on the presence of particular signature sequences. So we performed a text search of the EST database (dbEST) for precomputed annotation that included “tyrosine phosphatase.” GenBank AA527292 was one such EST
Discussion
Protein dephosphorylation by dual specificity phosphatases is an important mechanism for the regulation of cell signaling in a variety of systems. While most DSPs include an N-terminal domain that is important for substrate specificity and binding, a subclass of low molecular weight DSPs exists that lacks this domain. We have identified and characterized two novel LMW-DSPs. Both of these novel proteins contain the conserved PTP signature motif and catalytic domain but, like the LMW-DSP VHR [8],
Acknowledgements
We are grateful to Zhong-Yin Zhang for providing us with MKP-1 and MKP-3 constructs; Roger Davis for providing us with HA-ERK2, HA-JNK1, and HA-p38α; Michael Agostino for bioinformatics assistance and database searching; Laura Lin, Amy Tam, Tom McDonough, and Maria Lorenzo for protein purification; Lora Haines for DNA sequencing; and members of our laboratory for helpful discussion.
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Abbreviations: DSP, dual specificity phosphatase; EGF, epidermal growth factor; ERK, extracellular signal related kinase; EST, expressed sequence tag; JNK/SAPK, c-Jun NH2-terminal kinase/stress-activated protein kinase; JSP-1, JNK-stimulating protein-1; LMW, low molecular weight; MAPK, mitogen-activated protein kinase; MKP, MAP kinase phosphatase; PTP, protein tyrosine phosphatase; SKRP1, SAPK pathway-regulating phosphatase 1; VHR, VH1-related.