The very C-terminus of protein kinase Cepsilon is critical for the full catalytic competence but its hydrophobic motif is dispensable for the interaction with 3-phosphoinositide-dependent kinase-1.
In this article, we explore the role of the C-terminus (V5 domain) of PKCepsilon plays in the catalytic competence of the kinase using serial truncations followed by immune-complex kinase assays. Surprisingly, removal of the last seven amino acid residues at the C-terminus of PKCepsilon resulted in a PKCepsilon-Delta731 mutant with greatly reduced intrinsic catalytic activity while truncation of eight amino acid residues at the C-terminus resulted in a catalytically inactive PKCepsilon mutant. Computer modeling and molecular dynamics simulations showed that the last seven and/or eight amino acid residues of PKCepsilon were involved in interactions with residues in the catalytic core. Further truncation analyses revealed that the hydrophobic phosphorylation motif was dispensable for the physical interaction between PKCepsilon and 3-phosphoinositide-dependent kinase-1 (PDK-1) as the PKCepsilon mutant lacking both the turn and the hydrophobic motifs could still be co-immunoprecipitated with PDK-1. These results provide fresh insights into the biochemical and structural basis underlying the isozyme-specific regulation of PKC and suggest that the very C-termini of PKCs constitute a promising new target for the development of novel isozyme-specific inhibitors of PKC.
Zhu
Yimin
Y
Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Drive, 117597, Singapore.
Smith
Derek
D
Verma
Chandra
C
Lim
Wee Guan
WG
Tan
Bee Jen
BJ
Armstrong
Jeffrey S
JS
Zhou
Shufeng
S
Chan
Eli
E
Tan
Seng-Lai
SL
Zhu
Yi-Zhun
YZ
Cheung
Nam Sang
NS
Duan
Wei
W
D064413:3-Phosphoinositide-Dependent Protein Kinases; D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D003198:Computer Simulation; D017353:Gene Deletion; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D051744:Protein Kinase C-epsilon