Phosphorylation regulates the nucleocytoplasmic distribution of kinase suppressor of Ras.
KSR (kinase suppressor of Ras) has been proposed as a molecular scaffold regulating the Raf/MEK/ERK kinase cascade. KSR is phosphorylated on multiple phosphorylation sites by associated kinases. To identify potential mechanisms used by KSR to regulate ERK activation, green fluorescent protein was fused to intact and mutated KSR constructs lacking specific phosphorylation sites, and the subcellular distribution of each construct was observed in live cells. Mutation of a subset of KSR phosphorylation sites caused the redistribution of KSR to the nucleus. To determine whether intact KSR is normally imported to the nucleus, REF-52 fibroblasts expressing KSR were treated with 10 nm leptomycin B, which inhibits Crm1-dependent nuclear export. KSR accumulated in the nucleus within 2 h of treatment with leptomycin B, suggesting that KSR cycles continuously through the nucleus. Nuclear import of KSR was blocked by mutations that inhibit the interaction of KSR with MEK. Coexpression of fluorescent forms of KSR and MEK in cells revealed that each protein promoted the localization of the other in the cytoplasm. These data indicate that the subcellular distribution of KSR is dynamically regulated through phosphorylation and MEK interaction in a manner that may affect signaling through ERK.
Brennan
Jennifer A
JA
Eppley Institute for Research in Cancer and Allied Diseases, Department of Pathology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.
Volle
Deanna J
DJ
Chaika
Oleg V
OV
Lewis
Robert E
RE
D021581:Active Transport, Cell Nucleus; D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D005231:Fatty Acids, Unsaturated; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013912:Threonine; D012694:Serine; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D020928:Mitogen-Activated Protein Kinases; C038753:leptomycin B