Dynamic regulation of a metabolic multi-enzyme complex by protein kinase CK2

Abstract

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.

FIGURE 1.

Enzymes in human de novo purine biosynthesis. The de novo pathway transforms phosphoribosyl pyrophosphate (PRPP) to inosine monophosphate (IMP) in ten steps. Steps 2, 3, and 5 are catalyzed by a trifunctional enzyme TrifGART; steps 6 and 7 are catalyzed by a bifunctional enzyme PAICS; and steps 9 and 10 are catalyzed by a bifunctional enzyme ATIC.

FIGURE 2.

Chemical structures of small molecules that inhibit CK2 and Akt kinases.

FIGURE 3.

Effects of a CK2-specific inhibitor, TBI, on the purinosome formation in HeLa cells transiently expressing a GFP fusion protein. Representative cells expressing hFGAMS-GFP (a and b), hPPAT-GFP (c and d), or hTrifGART-GFP (e and f) were subjected to cellular imaging prior to addition of TBI (untreated: a, c, and e) and after the cells had been incubated with TBI for a given period (b, 30 min; d, 40 min; f, 60 min). The indicated regions of interest (white boxes) were enlarged for clarification. Scale bars, 10 μm.

FIGURE 4.

No alteration in cellular distribution of a non-purinosome member or non-CK2-substrate enzymes upon addition of TBI into singly transfected HeLa cells. Representative cells expressing hC1THF-GFP (a and b), hPAICS-GFP (c and d), human adenylosuccinate lyase (hASL)-GFP (e and f), or GFP-hATIC (g and h) were subjected to cellular imaging prior to addition of TBI (untreated: a, c, e, and g) and after the cells had been incubated with TBI for a given period (b, 60 min; d, 30 min; f, 30 min; and h, 50 min). Scale bars, 10 μm.

FIGURE 5.

No effect of an Akt-specific inhibitor, A-443654, added to the HeLa cells expressing hFGAMS-GFP. Representative cells grown in either purine-rich (a) or -depleted (c) medium were imaged prior to addition of A-443654, (untreated: a and c) and after the cells had been incubated with A-443654 for 70 min (b and d). The indicated region of interest (a white box) was enlarged for clarification. Scale bars, 10 μm.

FIGURE 6.

Reversibility of purinosome formation in HeLa cells by the sequential addition of DMAT and TBB. Representative cells expressing hFGAMS-GFP were monitored for purinosome formation prior to drug addition (a) and after incubating with DMAT for 25 min (b). DMAT-containing buffer was exchanged for fresh buffer containing TBB, subsequently promoting the dissociation of purinosomes within 20 min in the cells (c). Scale bars, 10 μm.

FIGURE 7.

Silencing the endogenous hCK2α catalytic subunit by RNA interference. Western blot analysis revealed that siCK2α-1 knocked down ∼45% of the expression level of the endogenous hCK2α catalytic subunit in HeLa cells (a and b). hCK2α expression was quantitatively normalized relative to the expression level of the endogenous hTrifGART (a and b). S.D. were indicated by error bars (b). In addition, cellular imaging experiments revealed that purinosomes were clearly detected in >95% of dually transfected HeLa cells coexpressing siCK2α-1 (c) and hFGAMS-OFP (d). Scale bars, 10 μm.