AHNAK-mediated activation of phospholipase C-gamma1 through protein kinase C.
We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by the central repeated units (CRUs) of the AHNAK protein in the presence of arachidonic acid. Here we demonstrate that four central repeated units (4 CRUs) of AHNAK act as a scaffolding motif networking PLC-gamma and PKC-alpha. Specifically, 4 CRUs of AHNAK bind and activate PKC-alpha, which in turn stimulates the release of arachidonic acid near where PLC-gamma1 is localized. Moreover, 4 CRUs of AHNAK interacted with PLC-gamma and the concerted action of 4 CRUs with arachidonic acid stimulated PLC-gamma activity. Stimulation of NIH3T3 cells expressing 4 CRUs of AHNAK with phorbol 12-myristate 13-acetate resulted in the increased generation of total inositol phosphates (IP(T)) and mobilization of the intracellular calcium. Phorbol 12-myristate 13-acetate-dependent generation of IP(T) was completely blocked in NIH3T3 cells depleted of PLC-gamma1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-beta isozyme resulting in the generation of a monophasic IP(T) within 30 s in NIH3T3 cells, led to a biphasic pattern for generation of IP(T) in NIH3T3 cells expressing 4 CRUs of AHNAK. The secondary activation of PLC is likely because of the scaffolding activity of AHNAK, which is consistent with the role of 4 CRUs as a molecular linker between PLC-gamma and PKC-alpha.
Lee
In Hye
IH
Division of Molecular Life Sciences, Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, Korea.
You
Je Ok
JO
Ha
Kwon Soo
KS
Bae
Duk Soo
DS
Suh
Pann-Ghill
PG
Rhee
Sue Goo
SG
Bae
Yun Soo
YS
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D001920:Bradykinin / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D041681:NIH 3T3 Cells; D009363:Neoplasm Proteins / Q000378:metabolism / Q000502:physiology*; D054799:Phospholipase C beta; D051966:Phospholipase C gamma; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry
C075176:AHNAK protein, human; C489572:Ahnak protein, mouse; D007295:Inositol Phosphates; D007527:Isoenzymes; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D016718:Arachidonic Acid; D014443:Tyrosine; D012694:Serine; D005982:Glutathione Transferase; D011493:Protein Kinase C; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D051966:Phospholipase C gamma; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D002118:Calcium