Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex.
We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A.
Lee
In Hye
IH
Department of Life Science, Ewha Womans University, 11-1 Daehyun-dong, Seodaemoon-Gu, Seoul 120-750, Korea.
Lim
Hee Jung
HJ
Yoon
Suhyeon
S
Seong
Je Kyung
JK
Bae
Duk Soo
DS
Rhee
Sue Goo
SG
Bae
Yun Soo
YS
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D051966:Phospholipase C gamma / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014280:Triglycerides / Q000378:metabolism; D048490:raf Kinases / Q000235:genetics / Q000378:metabolism
C489572:Ahnak protein, mouse; D002273:Carcinogens; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D010718:Phosphatidylserines; D014280:Triglycerides; C057439:1,3-dioleoyl-2-stearoylglycerol; D048490:raf Kinases; D051571:Protein Kinase C-alpha; D054648:Protein Phosphatase 2; D051966:Phospholipase C gamma; D013755:Tetradecanoylphorbol Acetate