Control of subcellular distribution of the MAP kinase phosphatase, MKP-2

Sloss, Callum

Thesis

Control of subcellular distribution of the MAP kinase phosphatase, MKP-2

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Creator

Sloss, Callum

Rights statement

Strathclyde Thesis Copyright

Awarding institution

University of Strathclyde

Date of award

2002

Thesis identifier

T10826

Qualification Level

Doctoral (Postgraduate)

Qualification Name

Doctor of Philosophy (PhD)

Department, School or Faculty

Abstract

Mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that play a key role in the transduction of signals from the cell membrane to the nucleus. MAP kinase activation is controlled by phosphorylation upon a T-X-Y (activation) motif. Inactivation of MAP kinases is achieved by dephosphorylation of the activation motif by a family of enzymes termed the MAP kinase phosphatases (MKPs). The MAP kinases are able to move from compartment to compartment whereas the phosphatases are predominantly restricted to either the cytosol or the nucleus. In this thesis the substrate specificity and the factors controlling the localisation of MKP-2 have been studied. Human MKP-2 was cloned from human umbilical vein endothelial cell cDNA library using PCR. A transient transfection system and epitope-tagged MAP kinases were then utilised to study the substrate specificity of human MKP-2 towards the MAP kinase family. MKP-2 was able to reverse JNK activation in COS7 cells, but unable to reduce either ERK or p38 MAPK activation. Creation of a catalytically inactive MKP-2 by mutation of the cysteine residue critical for phosphatase activity yielded a protein with no phosphatase activity that is able to act in a dominant negative fashion by preventing the activity of wild type MKP-2 towards JNK. Sequence analysis followed by mutational studies indicated two sequences within MKP-2 that are critical for subcellular localisation. Expression of single and double nuclear localisation sequence (NLS) mutants, defined NLS1 and NLS2 as novel localisation sequences, each individually able to direct MKP-2 to the nucleus. Loss of both NLS resulted in the cytosolic accumulation of MKP-2. The ability of nuclear export sequences derived from a cytosolic dual specificity phosphatase (DSP) to alter the locale of MKP-2 was also assessed. Addition of single nuclear export sequence (NES) proved insufficient to change the locale of MKP-2, suggesting that a single NES is unable to compete with two NLS.

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