Both nuclear-cytoplasmic shuttling of the dual specificity phosphatase MKP-3 and its ability to anchor MAP kinase in the cytoplasm are mediated by a conserved nuclear export signal.
MAP kinase phosphatase (MKP)-3 is a cytoplasmic dual specificity protein phosphatase that specifically binds to and inactivates the ERK1/2 MAP kinases in mammalian cells. However, the molecular basis of the cytoplasmic localization of MKP-3 or its physiological significance is unknown. We have used MKP-3-green fluorescent protein fusions in conjunction with leptomycin B to show that the cytoplasmic localization of MKP-3 is mediated by a chromosome region maintenance-1 (CRM1)-dependent nuclear export pathway. Furthermore, the nuclear translocation of MKP-3 seen in the presence of leptomycin B is mediated by an active process, indicating that MKP-3 shuttles between the nucleus and cytoplasm. The amino-terminal noncatalytic domain of MKP-3 is both necessary and sufficient for nuclear export of the phosphatase and contains a single functional leucine-rich nuclear export signal (NES). Even though this domain of the protein also mediates the binding of MKP-3 to MAP kinase, we show that mutations of the kinase interaction motif which abrogate ERK2 binding do not affect MKP-3 localization. Conversely, mutation of the NES does not affect either the binding or phosphatase activity of MKP-3 toward ERK2, indicating that the kinase interaction motif and NES function independently. Finally, we demonstrate that the ability of MKP-3 to cause the cytoplasmic retention of ERK2 requires both a functional kinase interaction motif and NES. We conclude that in addition to its established function in the regulated dephosphorylation and inactivation of MAP kinase, MKP-3 may also play a role in determining the subcellular localization of its substrate. Our results reinforce the idea that regulatory proteins such as MKP-3 may play a key role in the spatio-temporal regulation of MAP kinase activity.
Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, United Kingdom.
Maria
M
Karlsson
Joanne
J
Mathers
Robin J
RJ
Dickinson
Margret
M
Mandl
Stephen M
SM
Keyse
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D003593:Cytoplasm / Q000737:chemistry; D054642:Dual Specificity Phosphatase 6; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D019913:Nuclear Localization Signals; D010749:Phosphoprotein Phosphatases; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism* / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein
D054642:Dual Specificity Phosphatase 6; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D020928:Mitogen-Activated Protein Kinases; D019913:Nuclear Localization Signals; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C517035:DUSP6 protein, human; C517036:Dusp6 protein, mouse