PMID- 10373510
OWN - NLM
STAT- MEDLINE
DCOM- 19990722
LR  - 20190508
IS  - 0270-7306 (Print)
IS  - 0270-7306 (Linking)
VI  - 19
IP  - 7
DP  - 1999 Jul
TI  - Ras-specific exchange factor GRF: oligomerization through its Dbl homology domain
      and calcium-dependent activation of Raf.
PG  - 4611-22
AB  - The full-length versions of the Ras-specific exchange factors Ras-GRF1 (GRF1) and
      Ras-GRF2 (GRF2), which are expressed in brain and a restricted number of other
      organs, possess an ionomycin-dependent activation of Erk mitogen-activated
      protein kinase activity in 293T cells (C. L. Farnsworth et al., Nature
      376:524-527, 1995; N. P. Fam et al., Mol. Cell. Biol. 17:1396-1406, 1996). Each
      GRF protein contains a Dbl homology (DH) domain. A yeast two-hybrid screen was
      used to identify polypeptides that associate with the DH domain of GRF1. In this 
      screen, a positive cDNA clone from a human brain cDNA library was isolated which 
      consisted of the GRF2 DH domain and its adjacent ilimaquinone domain. Deletion
      analysis verified that the two-hybrid interaction required only the DH domains,
      and mutation of Leu-263 to Gln (L263Q) in the N terminus of the GRF1 DH domain
      abolished the two-hybrid interaction, while a cluster of more C-terminally
      located mutations in the DH domain did not eliminate the interaction. Oligomers
      between GRF1 and GRF2 were detected in a rat brain extract, and forced expression
      of GRF1 and GRF2 in cultured mammalian cells formed homo- and hetero-oligomers.
      Introduction of the L263Q mutation in GRF1 led to a protein that was deficient in
      oligomer formation, while GRF1 containing the DH cluster mutations formed
      homo-oligomers with an efficiency similar to that of wild type. Compared to
      wild-type GRF1, the focus-forming activity on NIH 3T3 cells of the GRF1 DH
      cluster mutant was reduced, while the L263Q mutant was inactive. Both mutants
      were impaired in their ability to mediate ionomycin-dependent Erk activity in
      293T cells. In the absence of ionomycin, 293T cells expressing wild-type GRF1
      contained much higher levels of Ras-GTP than control cells; the increase in Erk
      activity induced by ionomycin in the GRF1-expressing cells also induced a
      concomitant increase in Raf kinase activity, but without a further increase in
      the level Ras-GTP. We conclude that GRF1 and GRF2 can form homo- and
      hetero-oligomers via their DH domains, that mutational inactivation of oligomer
      formation by GRF1 is associated with impaired biological and signaling
      activities, and that in 293T cells GRF1 mediates at least two pathways for Raf
      activation: one a constitutive signal that is mainly Ras-dependent, and one an
      ionomycin-induced signal that cooperates with the constitutive signal without
      further augmenting the level of GTP-Ras.
FAU - Anborgh, P H
AU  - Anborgh PH
AD  - Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland
      20892, USA.
FAU - Qian, X
AU  - Qian X
FAU - Papageorge, A G
AU  - Papageorge AG
FAU - Vass, W C
AU  - Vass WC
FAU - DeClue, J E
AU  - DeClue JE
FAU - Lowy, D R
AU  - Lowy DR
LA  - eng
SI  - GENBANK/AF023130
PT  - Journal Article
PL  - United States
TA  - Mol Cell Biol
JT  - Molecular and cellular biology
JID - 8109087
RN  - 0 (DNA, Complementary)
RN  - 0 (Guanine Nucleotide Exchange Factors)
RN  - 0 (Ionophores)
RN  - 0 (Oligopeptides)
RN  - 0 (Proteins)
RN  - 0 (ras Guanine Nucleotide Exchange Factors)
RN  - 0 (ras-GRF1)
RN  - 56092-81-0 (Ionomycin)
RN  - EC 2.7.11.1 (Proto-Oncogene Proteins c-raf)
RN  - EC 2.7.11.17 (Calcium-Calmodulin-Dependent Protein Kinases)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - 3T3 Cells
MH  - Amino Acid Sequence
MH  - Animals
MH  - Binding Sites
MH  - Brain/metabolism
MH  - COS Cells
MH  - Calcium/*metabolism
MH  - Calcium-Calmodulin-Dependent Protein Kinases/metabolism
MH  - Cell Line, Transformed
MH  - Chromosome Mapping
MH  - Cloning, Molecular
MH  - DNA, Complementary
MH  - Enzyme Activation
MH  - Guanine Nucleotide Exchange Factors
MH  - Humans
MH  - Ionomycin/pharmacology
MH  - Ionophores/pharmacology
MH  - Mice
MH  - Molecular Sequence Data
MH  - Oligopeptides/genetics/metabolism
MH  - Proteins/genetics/*metabolism
MH  - Proto-Oncogene Proteins c-raf/*metabolism
MH  - Rats
MH  - Sequence Homology, Amino Acid
MH  - ras Guanine Nucleotide Exchange Factors
MH  - ras-GRF1
PMC - PMC84259
EDAT- 1999/06/22 00:00
MHDA- 1999/06/22 00:01
CRDT- 1999/06/22 00:00
PHST- 1999/06/22 00:00 [pubmed]
PHST- 1999/06/22 00:01 [medline]
PHST- 1999/06/22 00:00 [entrez]
AID - 10.1128/mcb.19.7.4611 [doi]
PST - ppublish
SO  - Mol Cell Biol. 1999 Jul;19(7):4611-22. doi: 10.1128/mcb.19.7.4611.