Loop 6 of RhoA confers specificity for effector binding, stress fiber formation, and cellular transformation

Hui Zong, Narayan Raman, Leigh A. Mickelson-Young, Simon J. Atkinson, Lawrence A. Quilliam

Research output: Contribution to journalArticle

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Abstract

Rho family GTPases regulate multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These effects are achieved through the interaction of GTP-bound proteins with various downstream targets. A series of RhoA/Rac1 and Rho/Ras chimeras was generated to map the domain(s) of RhoA involved in its association with two classes of effector kinase, represented by PRK2 and ROCK-I. Although the switch 1 domain was required for effector binding, the N terminus of Rho (residues 1-75) was interchangeable with that of Rac. This suggested that the region of Rho that confers effector binding specificity lay further C-terminal. Subsequent studies indicated that the 'insert domain'(residues 123-137), a region unique to Rho family GTPases, is not the specificity determinant. However, a determinant for effector binding was identified between Rho residues 75-92. Rac to Rho point mutations (V85D or A88D) within loop 6 of Rac promoted its association with PRK2 and ROCK, whereas the reciprocal Rho(D87V/D90A) double mutant significantly reduced effector binding capacity. In vivo studies showed that microinjection of Rac(Q6IL/V85D/A88D) but not Rac(Q6IL) induced stress fiber formation in LLC-PK epithelial cells, suggesting that loop 6 residues conferred the ability of Rac to activate ROCK. On the other hand, the reciprocal Rho (Q6IL/D87V/D90A) mutant was defective in its ability to transform NIH 3T3 cells. These data suggest that although Rho effectors can utilize a Rho or Rac switch 1 domain to sense the GTP-bound state of Rho, unique residues within loop 6 are essential for determining both effector binding specificity and cellular function.

Original languageEnglish (US)
Pages (from-to)4551-4560
Number of pages10
JournalJournal of Biological Chemistry
Volume274
Issue number8
DOIs
StatePublished - Feb 19 1999

Fingerprint

Stress Fibers
rho GTP-Binding Proteins
GTP Phosphohydrolases
Guanosine Triphosphate
Switches
NIH 3T3 Cells
Fibers
Microinjections
Point Mutation
Gene expression
Epithelial Cells
Gene Expression
Proteins
protein kinase N

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Loop 6 of RhoA confers specificity for effector binding, stress fiber formation, and cellular transformation. / Zong, Hui; Raman, Narayan; Mickelson-Young, Leigh A.; Atkinson, Simon J.; Quilliam, Lawrence A.

In: Journal of Biological Chemistry, Vol. 274, No. 8, 19.02.1999, p. 4551-4560.

Research output: Contribution to journalArticle

Zong, Hui ; Raman, Narayan ; Mickelson-Young, Leigh A. ; Atkinson, Simon J. ; Quilliam, Lawrence A. / Loop 6 of RhoA confers specificity for effector binding, stress fiber formation, and cellular transformation. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 8. pp. 4551-4560.
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