Mechanism of inhibition of Raf-1 by protein kinase A.
The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.
Häfner
S
S
Institut für Klinische Molekularbiologie und Tumorgenetik, Munich.
Adler
H S
HS
Mischak
H
H
Janosch
P
P
Heidecker
G
G
Wolfman
A
A
Pippig
S
S
Lohse
M
M
Ueffing
M
M
Kolch
W
W
D016475:3T3 Cells; D000818:Animals; D002471:Cell Transformation, Neoplastic; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D048728:MAP Kinase Kinase Kinase 1; D051379:Mice; D027384:Oncogene Proteins v-raf; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D015682:Retroviridae Proteins, Oncogenic; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D027384:Oncogene Proteins v-raf; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D048728:MAP Kinase Kinase Kinase 1; C482578:Map3k1 protein, mouse; D016283:Proto-Oncogene Proteins p21(ras)