Activation loop phosphorylation-independent kinase activity of human protein kinase C zeta.
Atypical protein kinase C zeta (PKCzeta) plays an important role in cell proliferation and survival. PKCzeta and its truncated form containing only the kinase domain, CATzeta, have been reported to be activated by the phosphorylation of threonine 410 in the activation loop. We expressed both the full length PKCzeta and CATzeta in a baculovirus/insect cell over-expression system and purified the proteins for biochemical characterization. Ion exchange chromatography of CATzeta revealed three species with different levels of phosphorylation at Thr-410 and allowed the isolation of the CATzeta protein devoid of phosphorylation at Thr-410. All three species of CATzeta were active and their activity was not correlated with phosphorylation at Thr-410, indicating that the kinase activity of CATzeta did not depend solely on activation loop phosphorylation. Tyrosine phosphorylation was detected in all three species of CATzeta and the full length PKCzeta. Homology structural modeling of PKCzeta revealed a conserved, predicted-to-be phosphorylated tyrosine residue, Tyr-428, in the close proximity of the RD motif of the catalytic loop and of Thr-410 in the activation loop. The structural analysis indicated that phospho-Tyr-428 would interact with two key, positively-charged residues to form a triad conformation similar to that formed by phospho-Thr-410. Based on these observations, it is possible that the Thr-410 phosphorylation-independent kinase activity of CATzeta is regulated by the phosphorylation of Tyr-428. This alternative mode of PKCzeta activation is supported by the observed stimulation of PKCzeta kinase activity upon phosphorylation at the equivalent site by Abl, and may be involved in resistance to drug-induced apoptosis.
Ranganathan
Senthil
S
Department of Organic Chemistry, Drug Discovery Division, Southern Research Institute, 2000, 9th Avenue South, Birmingham, AL 35205, USA.
Wang
Yimin
Y
Kern
Francis G
FG
Qu
Zhican
Z
Li
Rongbao
R
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D020134:Catalytic Domain; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013329:Structure-Activity Relationship; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D000099221:Protein Kinase C zeta
D064413:3-Phosphoinositide-Dependent Protein Kinases; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D013912:Threonine; D014443:Tyrosine; D000099221:Protein Kinase C zeta