Faculty Collaboration Database

Unique in vivo associations with SmgGDS and RhoGDI and different guanine nucleotide exchange activities exhibited by RhoA, dominant negative RhoA(Asn-19), and activated RhoA(Val-14). J Biol Chem 2000 Mar 10;275(10):6699-702

Date

03/04/2000

Pubmed ID

10702222

DOI

10.1074/jbc.275.10.6699

Scopus ID

2-s2.0-0034629117 (requires institutional sign-in at Scopus site)   24 Citations

Abstract

We compared the in vivo characteristics of hemagglutinin (HA)-tagged RhoA, dominant negative RhoA(Asn-19), and activated RhoA(Val-14) stably expressed in Chinese hamster ovary (CHO) cells. Proteins co-precipitating with these HA-tagged GTPases were identified by peptide sequencing or by Western blotting. Dominant negative RhoA(Asn-19) co-precipitates with the guanine nucleotide exchange factor (GEF) SmgGDS but does not detectably interact with other expressed GEFs, such as Ost or Dbl. SmgGDS co-precipitates minimally with wild-type RhoA and does not detectably associate with RhoA(Val-14). The guanine nucleotide dissociation inhibitor RhoGDI co-precipitates with RhoA, and to a lesser extent with RhoA(Val-14), but does not detectably co-precipitate with RhoA(Asn-19). Wild-type RhoA is predominantly in the [(32)P]GDP-bound form, RhoA(Val-14) is predominantly in the [(32)P]GTP-bound form, and negligible levels of [(32)P]GDP or [(32)P]GTP are bound to RhoA(Asn-19) in (32)P-labeled cells. Immunofluorescence analyses indicate that HA-RhoA(Asn-19) is excluded from the nucleus and cell junctions. Microinjection of SmgGDS cDNA into CHO cells stably expressing HA-RhoA causes HA-RhoA to be excluded from the nucleus and cell junctions, similar to the distribution of RhoA(Asn-19). Our findings indicate that the expression of RhoA(Asn-19) may specifically inhibit signaling pathways that rely upon the SmgGDS-dependent activation of RhoA.

Author List

Strassheim D, Porter RA, Phelps SH, Williams CL

Author

Carol L. Williams PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Animals
CHO Cells
Cricetinae
Guanine Nucleotide Dissociation Inhibitors
Guanine Nucleotide Exchange Factors
Guanosine Diphosphate
Guanosine Triphosphate
Molecular Sequence Data
rho Guanine Nucleotide Dissociation Inhibitor alpha
rho-Specific Guanine Nucleotide Dissociation Inhibitors
rhoA GTP-Binding Protein