Mutations in ERK2 binding sites affect nuclear entry.
The MAPK ERK2 can enter and exit the nucleus by an energy-independent process that is facilitated by direct interactions with nuclear pore proteins. Several studies also suggest that the localization of ERK2 can be influenced by carrier proteins. Using import reconstitution assays, we examined a group of ERK2 mutants defective in known protein interactions to determine structural properties of ERK2 that contribute to its nuclear entry. ERK2 mutants defective in binding to substrates near the active site or to basic/hydrophobic docking (D) motifs were imported normally. Several ERK2 mutants defective in interactions with FXF motifs displayed slowed rates of nuclear import. The import-impaired mutants also showed reduced binding to a recombinant C-terminal fragment of nucleoporin 153 that is rich in FXF motifs. Despite the deficit revealed in some mutants via reconstitution assays, all but one of the ERK2 mutants accumulated in nuclei of stimulated cells in a manner comparable with the wild type protein; the mutant most defective in import remained in the cytoplasm. These results further support the idea that direct interactions with nucleoporins are involved in ERK2 nuclear entry and that multiple events contribute to the ligand-dependent relocalization of these protein kinases.
Departments of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041.
Mustafa N
MN
Yazicioglu
Departments of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041.
Daryl L
DL
Goad
Departments of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041.
Aarati
A
Ranganathan
Departments of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041.
Angelique W
AW
Whitehurst
Departments of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041.
Elizabeth J
EJ
Goldsmith
Departments of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9041. Electronic address: Melanie.Cobb@UTSouthwestern.edu.
Melanie H
MH
Cobb
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D028861:Nuclear Pore Complex Proteins; C513083:Nup153 protein, rat; D011994:Recombinant Proteins; D019950:Mitogen-Activated Protein Kinase 1