MULTIPLE INTERACTIONS OF PRK1 WITH RHOA - FUNCTIONAL ASSIGNMENT OF THE HR1 REPEAT MOTIF

PRK1 (PKN) is a serine/threonine kinase that has been shown to be acti vated by RhoA (Amano, M., Mukai, H., One, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Scienc e 271, 648-650), Detailed analysis of the PRK1 region involved in RhoA binding has revealed that two homologous sequences within the HR1 dom ain (HR1a and HR1b) both bind to RhoA; the third repeat within this do main, HR1c(PRK1), does not bind RhoA, The related HR1 motif is also fo und to confer RhoA binding activity to the only other fully cloned mem ber of this kinase family, PRK2, Furthermore, the predictive value of this motif is established for an HR1a sequence derived from a Caenorha bditis elegans open reading frame encoding a protein kinase of unknown function, Interestingly, the HR1a(PRK1) and HR1b(PRK1) subdomains are shown to display a distinctive nucleotide dependence for RhoA binding , HR1a(PRK1) is entirely GTP-dependent, while HR1b(PRK1) binds both GT P-and GDP-bound forms of RhoA. This distinction indicates that there a re two sites of contact between RhoA and PRK1, one contact through a r egion that is conformationally dependent upon the nucleotide-bound sta te of RhoA and one that is not, Analysis of binding to Rho/Rac chimera provides evidence for a HR1a(PRK1) but not HR1b(PRK1) interaction in the central third of Rho, Additionally, it is observed that the V14Rho A mutant binds HR1a but does not bind HR1b, This distinct binding beha vior corroborates the conclusion that there are independent contacts o n RhoA for the HR1a(PRK1) and HR1b(PRK1) motifs.