Disruption of two putative nuclear localization sequences is required for cytosolic localization of mitogen-activated protein kinase phosphatase-2
Introduction
The mitogen-activated protein (MAP) kinases are a family of proteins that consist of the classical p42/44 MAP kinases or extracellular-signal regulated kinases (ERKs), the homologues of the stress-activated protein kinases c-Jun N-terminal kinase (JNK) and p38 MAP kinase and also big MAPK [1], [2], [3], [4]. These kinases play an important role in the regulation of cell growth and division by regulating a number of transcription factors, such as AP-1, ELK-1, c-Jun and CHOP [3], [5], [6], [7]. Recent studies have shown that MAP kinase homologues have both cytosolic and nuclear functions, suggesting that subcellular distribution is a key feature of their actions.
MAP kinase phosphatase-2 (MKP-2) is a dual specificity phosphatase (DSP) found in a wide variety of tissues and a member of a much larger family of DSPs that are known to dephosphorylate and inactivate MAP kinase homologues (for review, see Ref. [8]). Each MKP is unique in its tissue distribution and its substrate specificity amongst the MAP kinase signaling cascade superfamily. For example, the substrate specificity of the nuclear-located MKP-2 has been identified as being ERK and JNK [9]. This is in contrast to another nuclear DSP, MKP-1, which can dephosphorylate ERK, JNK and p38 MAP kinase [9], [10] or the cytosolic DSP, MKP-3 which specifically dephosphorylates ERK alone [11], [12].
Given that the subcellular distribution of MKP-2 is important in defining its function, we sought to determine the sequences involved in the nuclear targeting of the protein. To date, very little is known regarding the control of subcellular localization of the MKPs at the structural level. Their distribution is thought to be dependent on the presence of NLS and nuclear export sequences (NES), which have been shown to control the subcellular distribution of many proteins [13]. Indeed, putative NLS and NES have been identified in several members of the MKP family [14], [15].
In this study, we analyzed the role of two putative NLS on the control of MKP-2 localization. We find that disruption of both putative localization sequences is required for redistribution of MKP-2 to the cytosol and that expression of either NLS alone is sufficient to target the protein to the nucleus. Disruption of both sequences prevented the inactivation of JNK activity due to differential compartmentalization of the phosphatase.
Section snippets
Materials
All materials used were of the highest commercial grade available and were obtained from Sigma, Poole, Dorset, UK unless otherwise stated.
The GST-tagged truncated N-terminus c-Jun5–89 was a kind gift of J.R. Woodgett (Ontario Cancer Institute, Toronto, Canada). The atrial natriuretic factor-luciferase (ANF-LUX) reporter vector, pANF(-3003)LΔ5′ [16] and an expression vector (pON249), in which the β-galactosidase (β-gal) gene is under the control of the human CMV promoter [17] were provided by
Localization of MKP-2
As described above, the MKPs are predominantly either cytosolic or nuclear and the mechanism which defines this subcellular distribution is unclear. Fig. 1 is a schematic of MKP-2 showing the two putative NLS that are thought to be responsible for its subcellular distribution, and the phosphatase catalytic domain. Although no definitive consensus sequence allowing easy identification of NLS has been found, they tend to contain short sections of basic sequence (usually arginine and lysine) [22]
Discussion
In this study, we sought to identify regions within MKP-2, which are responsible for nuclear targeting of the protein. We have identified two NLS, Both of these participate in the localization of the protein and the presence of either is sufficient to retain MKP-2 within the nucleus. The first of these putative sequences (NLS-1) is similar to the prototypic NLS motif identified in a number of nuclear proteins, including SV40 large T-antigen [26]. NLS-2 is similar to a different type of NLS, a
Acknowledgment
This work was sponsored by a BBSRC grant to R.P. and BHF grant PG/98131 to S.J.F. Callum M. Sloss is an MRC quota award student.
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