Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts.
Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.
Centre de Biochimie-CNRS, Université de Nice, France.
P
P
Lenormand
C
C
Sardet
G
G
Pagès
G
G
L'Allemain
A
A
Brunet
J
J
Pouysségur
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001692:Biological Transport / Q000502:physiology; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D006224:Cricetinae; D003593:Cytoplasm / Q000201:enzymology / Q000648:ultrastructure; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006133:Growth Substances / Q000494:pharmacology*; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D007536:Isomerism; D008168:Lung / Q000166:cytology; D020929:Mitogen-Activated Protein Kinase Kinases; D008934:Mitogens / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010703:Phorbol Esters / Q000494:pharmacology; D011233:Precipitin Tests; D011494:Protein Kinases / Q000032:analysis / Q000276:immunology / Q000493:pharmacokinetics*; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D006133:Growth Substances; D007106:Immune Sera; D008934:Mitogens; D010703:Phorbol Esters; D004247:DNA; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D013917:Thrombin