Epidermal growth factor receptor and protein kinase C signaling to ERK2: spatiotemporal regulation of ERK2 by dual specificity phosphatases.
Spatiotemporal aspects of ERK activation are stimulus-specific and dictate cellular consequences. They are dependent upon dual specificity phosphatases (DUSPs) that bind ERK via docking domains and can both inactivate and anchor ERK in cellular compartments. Using high throughput fluorescence microscopy in combination with a system where endogenous ERKs are removed and replaced with wild-type or mutated ERK2-green fluorescent protein (GFP), we show that ERK2 activation responses to epidermal growth factor (EGF) and protein kinase C (PKC) are transient and sustained, respectively. PKC-mediated ERK2 activation is associated with prolonged nuclear localization in the dephosphorylated form, whereas EGF-stimulated ERK2 activation mediates only transient nuclear accumulation. By using short inhibitory RNAs to nuclear inducible DUSP1, -2, or -4 (alone or in combination), we demonstrate that all three of these enzymes contribute to the dephosphorylation of PKC (but not EGF)-activated ERK2 in the nucleus but that they have opposing effects on localization. DUSP2 and -4 inactivate and anchor ERK2, whereas DUSP1 dephosphorylates ERK in the nucleus but allows its traffic back to the cytoplasm. Overexpression of DUSP1, -2, or -4 prevented ERK2 activation, but only DUSP2 and -4 caused ERK2-GFP nuclear accumulation or could be immunoprecipitated with ERK2. Furthermore, protein synthesis inhibition or replacement of wild-type ERK2-GFP with docking domain mutants selectively increased PKC effects on ERK activity and altered ERK2-GFP localization. These mutations also impaired the ability of ERK2-GFP to bind DUSP2 and -4. Together, our data reveal a novel, stimulus-specific, and phosphatase-specific mechanism of ERK2 regulation in the nucleus by DUSP1, -2, and -4.
Caunt
Christopher J
CJ
Laboratories for Integrated Neuroscience and Endocrinology, Department of Clinical Sciences at South Bristol, University of Bristol, Whitson Street, Bristol, U.K.
Rivers
Caroline A
CA
Conway-Campbell
Becky L
BL
Norman
Michael R
MR
McArdle
Craig A
CA
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D002467:Cell Nucleus / Q000201:enzymology* / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation / Q000502:physiology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology
C512478:EGFR protein, human; D066246:ErbB Receptors; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D054639:Mitogen-Activated Protein Kinase Phosphatases