ERK1 nucleocytoplasmic shuttling rate depends on specific N-terminal aminoacids.
Despite ERK1 and ERK2 were considered interchangeable isoforms for a long time, their roles are now emerging as only partially overlapping. We recently reported that the nucleocytoplasmic trafficking of GFP-tagged ERK1 is slower than that of ERK2, this difference being caused by a unique domain of ERK1 located at its N-terminus (ERK1-Nt). In the present report we further investigated this issue by asking which were the specific aminoacids involved in such process. By photobleaching strategy, we demonstrated that ERK1-Nt is a domain capable to slow down the nucleocytoplasmic shuttling rate even of a small cargo protein. ERK1-Nt was then dissected into three regions as follows: 1 (aa 1-9), 2 (aa 10-29) and 3, (aa 30-39) that were deleted or mutated at specific sites. Dynamic imaging assessment of the role played by each region in determining the shuttling rate revealed that: region 1 has no significant role, region 2 and specific aminoacids of region 3 (V31, K33, P36) are critical, but singularly do not totally account for the difference in the shuttling rate between ERK1 and 2. Finally, we demonstrated that the nucleocytoplasmic shuttling rate of a passively diffusing protein (mRED) is inversely related to ERK1-Nt-GFP concentrations inside the cell, thus suggesting that ERK1-Nt-GFP occupies the nuclear pore perhaps because of an important affinity of ERK1-Nt for nucleoporins. In conclusion, ERK1-Nt is a domain able per se to confer a slower shuttling rate to a cargo protein. Specific regions within this domain were identified as responsible for this biophysical property.
Marchi
Matilde
M
Dulbecco Telethon Institute, Department of Endocrinology and Kidney, University Hospital of Pisa,and NEST-INFM, Scuola Normale Superiore, Pisa, Italy.
Pancrazi
Laura
L
Maffei
Margherita
M
Ratto
G Michele
GM
Costa
Mario
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D041681:NIH 3T3 Cells; D021381:Protein Transport / Q000235:genetics; D051381:Rats
D000596:Amino Acids; D049452:Green Fluorescent Proteins; D048052:Mitogen-Activated Protein Kinase 3