The C-terminus of PRK2/PKNgamma is required for optimal activation by RhoA in a GTP-dependent manner.
PRK2/PKNgamma is a Rho effector and a member of the protein kinase C superfamily of serine/threonine kinases. Here, we explore the structure-function relationship between various motifs in the C-terminal half of PRK2 and its kinase activity and regulation. We report that two threonine residues at conserved phosphoacceptor position in the activation loop and the turn motif are essential for the catalytic activity of PRK2, but the phosphomimetic Asp-978 at hydrophobic motif is dispensable for kinase catalytic competence. Moreover, the PRK2-Delta958 mutant with the turn motif truncated still interacts with 3-phosphoinositide-dependent kinase-1 (PDK-1). Thus, both the intact hydrophobic motif and the turn motif in PRK2 are dispensable for the binding of PDK-1. We also found that while the last seven amino acid residues at the C-terminus of PRK2 are not required for the activation of the kinase by RhoA in vitro, however, the extreme C-terminal segment is critical for the full activation of PRK2 by RhoA in cells in a GTP-dependent manner. Our data suggest that the extreme C-terminus of PRK2 may represent a potential drug target for effector-specific pharmacological intervention of Rho-medicated biological processes.
Department of Biochemistry, Yong Loo Lin of Medicine, The National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore.
Wee Guan
WG
Lim
Xiao
X
Chen
Jun-Ping
JP
Liu
Bee Jen
BJ
Tan
Shufeng
S
Zhou
Adam
A
Smith
Nathaniel
N
Lees
Liansheng
L
Hou
Fukang
F
Gu
Xi Yong
XY
Yu
Yaomin
Y
Du
Derek
D
Smith
Chandra
C
Verma
Ke
K
Liu
Wei
W
Duan
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; C066737:RHOA protein, human; C061570:protein kinase N; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D020742:rhoA GTP-Binding Protein