Interactions of the amino acid residue at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS.
The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.
Shirouzu
M
M
Cellular Signaling Laboratory, Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, Japan.
Morinaka
K
K
Koyama
S
S
Hu
C D
CD
Hori-Tamura
N
N
Okada
T
T
Kariya
K
K
Kataoka
T
T
Kikuchi
A
A
Yokoyama
S
S
D016475:3T3 Cells; D000818:Animals; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D016716:PC12 Cells; D017354:Point Mutation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship; D020726:ral Guanine Nucleotide Exchange Factor; D020668:rap GTP-Binding Proteins; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020726:ral Guanine Nucleotide Exchange Factor; D018698:Glutamic Acid; D011494:Protein Kinases; D019908:Proto-Oncogene Proteins c-raf; D020929:Mitogen-Activated Protein Kinase Kinases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D018631:ras Proteins