Dynamic regulation of a metabolic multi-enzyme complex by protein kinase CK2.
The reversible association and dissociation of a metabolic multi-enzyme complex participating in de novo purine biosynthesis, the purinosome, was demonstrated in live cells to respond to the levels of purine nucleotides in the culture media. We also took advantage of in vitro proteomic scale studies of cellular substrates of human protein kinases (e.g. casein kinase II (CK2) and Akt), that implicated several de novo purine biosynthetic enzymes as kinase substrates. Here, we successfully identified that purinosome formation in vivo was significantly promoted in HeLa cells by the addition of small-molecule CK2-specific inhibitors (i.e. 4,5,6,7-tetrabromo-1H-benzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, tetrabromocinammic acid, 4,4',5,5',6,6'-hexahydroxydiphenic acid 2,2',6,6'-dilactone (ellagic acid) as well as by silencing the endogenous human CK2alpha catalytic subunit with small interfering RNA. However, 4,5,6,7-tetrabromobenzotriazole, another CK2-specific inhibitor, triggered the dissociation of purinosome clusters in HeLa cells. Although the mechanism by which 4,5,6,7-tetrabromobenzotriazole affects purinosome clustering is not clear, we were capable of chemically reversing purinosome formation in cells by the sequential addition of two CK2 inhibitors. Collectively, we provide compelling cellular evidence that CK2-mediated pathways reversibly regulate purinosome assembly, and thus the purinosome may be one of the ultimate targets of kinase inhibitors.
Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Songon
S
An
Minjoung
M
Kyoung
Jasmina J
JJ
Allen
Kevan M
KM
Shokat
Stephen J
SJ
Benkovic
D047390:Casein Kinase II / Q000378:metabolism / Q000502:physiology*; D002384:Catalysis; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D008956:Models, Chemical; D011687:Purines / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism
D004791:Enzyme Inhibitors; D011687:Purines; D034741:RNA, Small Interfering; D047390:Casein Kinase II; C030985:purine