Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S in CD3-stimulated primary human T cells.
To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication.
Zmuidzinas
A
A
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Mamon
H J
HJ
Roberts
T M
TM
Smith
K A
KA
D015703:Antigens, CD / Q000502:physiology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology*; D015152:Blotting, Northern; D015153:Blotting, Western; D017252:CD3 Complex; D002453:Cell Cycle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003524:Cyclosporins / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D010766:Phosphorylation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D011519:Proto-Oncogenes; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D016192:Resting Phase, Cell Cycle; D016196:S Phase; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects*
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D003524:Cyclosporins; D007376:Interleukin-2; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D012313:RNA; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; D019908:Proto-Oncogene Proteins c-raf