Mitogenic signaling of Ras is regulated by differential interaction with Raf isozymes.
In the mitogenic signaling cascade interaction of Ras with Raf represents a critical step for the regulation of cell growth and differentiation. The major effector of Ras, the serine/threonine kinase Raf exists as three isoforms with different tissue distributions. We demonstrate that transient transfection of oncogenic Ha-Ras leads to a preferential activation of endogenous c-Raf-1 in HEK 293 cells as opposed to A-Raf. In vitro binding studies using purified Ras binding domains of Raf as well as in vivo bindings tests with full length molecules reveals significantly lower binding affinities of A-Raf to Ha-Ras as compared to other Raf isoforms. The Ras-binding interface of c-Raf differs from A-Raf by a conservative Arg to Lys exchange at residue 59 or 22 respectively. Mutational analysis reveals that this residue represents a point of isozyme discrimination: c-Raf-R59K binds Ha-Ras weaker than the wildtype, likewise A-Raf-K22R increases its affinity to Ha-Ras in vivo and in vitro. Differential binding affinities are reflected in downstream signaling. Immunecomplex kinase assays reveal that Ha-Ras mediated Raf activation is decreased for c-Raf-R59K and increased for A-Raf-K22R when compared to the respective wildtype forms. Thus our observations introduce a new level of isoform discrimination in Ras/Raf signaling as a functional consequence of a conservative amino acid exchange in the Ras binding domains.
Weber
C K
CK
Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Universität Würzburg, Versbacher Str.5, 97078 Würzburg, Germany.
Slupsky
J R
JR
Herrmann
C
C
Schuler
M
M
Rapp
U R
UR
Block
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008934:Mitogens / Q000502:physiology*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism / Q000502:physiology*; D011817:Rabbits; D013379:Substrate Specificity / Q000235:genetics; D014162:Transfection
D007527:Isoenzymes; D008934:Mitogens; D019908:Proto-Oncogene Proteins c-raf; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)