Isolation and Characterisation of a Uniquely Regulated Threonine, Tyrosine Phosphatase (TYP 1) Which Inactivates Mitogen-Activated Protein Kinases

King, Andrea Georgina (1995) Isolation and Characterisation of a Uniquely Regulated Threonine, Tyrosine Phosphatase (TYP 1) Which Inactivates Mitogen-Activated Protein Kinases. PhD thesis, University of Glasgow.

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The recent discovery of the vaccinia virus protein phosphatase VH1, and its mammalian counterparts has highlighted a novel subfamily of protein phosphatases that exhibit dual specificity towards phosphotyrosine- and phosphoserine/threonine- residues. The aim of this thesis was to isolate and characterise further members of this phosphatase subfamily. Employing the technique of polymerase chain reaction (PCR), degenerate PCR primers, designed from amino acid sequences conserved between two members of this dual specificity phosphatase family, were used to amplify related sequences from poly (A)+ RNA isolated from the human squamous cell line A431. Screening cDNA libraries with probes derived from the PCR analysis resulted in the isolation of five novel genes, which show significant homology to the catalytic domain of the dual specificity phosphatases, such as the human phosphatase CL100. Two of these, TYP 1 and TYP 2 (threonine-tyrosine phosphatase), have been analysed further. Interestingly, whilst TYP 2 expression is regulated with similar kinetics to CL100, TYP 1 differs dramatically in its regulation. The TYP 1 gene is not expressed in human fibroblasts unlike other CL100-like genes. Furthermore, northern analysis has demonstrated that following mitogenic stimulation of squamous cells, induction of TYP 1 mRNA reaches its maximal levels after four hours, in contrast to the immediate-early CL100-like genes. Both TYP 1 and CL100 mRNAs are induced upon transforming growth factor- beta treatment of squamous cell lines sensitive to the growth factors antiproliferative effects. Investigations were undertaken to determine if TYP 1 demonstrated substrate specificity towards the mitogen-activated protein kinases, a function peculiar to the other CL100-like phosphatases. Indeed, transient transfection of TYP 1 into COS-1 cells resulted in the inhibition of both ERK2 and p54JNK MAP kinase isoforms. In addition, purified TYP 1 protein efficiently inactivates recombinant ERK2 in vitro by the concomitant dephosphorylation of both the phosphothreonine and -tyrosine residues. The subcellular localisation of TYP 1 was investigated, and shown to encode a nuclear protein. Interestingly, the mitogenic induction of TYP 1 results in an increase in the half-life of the protein. The results presented in this thesis suggest that the dual specificity phosphatases are an expanding family, some members of which are differentially regulated, therefore providing alternative mechanisms for the control of cellular responses to different ligands in different cell types. As discussed, TYP 1 is particularly exciting as its kinetics of induction and cell specificity of expression make it unique in terms of its possible roles.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Brad Ozanne
Keywords: Molecular biology, Genetics
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-75803
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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