Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation.
Biliverdin reductase (BVR) reduces heme oxygenase (HO) activity product, biliverdin, to bilirubin. BVR is unique in having dual pH/dual cofactor requirements. Using Escherichia coli-expressed human BVR and COS cells, we show that BVR is autophosphorylated and that phosphorylation is required for its activity. An "in blot" autophosphorylation assay showed that BVR is a renaturable phosphoprotein. Controls for the experiments were HO-1 and HO-2; both are phosphoproteins but are not autophosphorylated. Autophosphorylation was pH-dependent, with activity at pH 8.7 being most prominent. In addition, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate fluorescence titration of BVR gave a lower K(d) at pH 8.7 than at pH 7.4 (15.5 versus 28.0 micrometer). Mn(2+) was required for binding of the ATP analogue and for autophosphorylation; the autokinase activity was lost when treated at 60 degrees C for 10 min. The loss of transferred phosphates by alkaline treatment suggested that BVR is a serine/threonine kinase. Potato acid phosphatase treatment reversibly inactivated the enzyme. The enzyme was also inactivated by treatment with the serine/threonine phosphatase, protein phosphatase 2A; okadaic acid attenuated the inhibition. Titration of protein phosphatase 2A-released phosphates indicated a 1:6 molar ratio of BVR to phosphate. The BVR immunoprecipitated from COS cell lysates was a phosphoprotein, and its activity and phosphorylation levels increased in response to H(2)O(2). The results define a previously unknown mechanism for regulation of BVR activity and are discussed in the context of their relevance to heme metabolism.
Department of Biochemistry/Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
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M
Salim
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BA
Brown-Kipphut
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MD
Maines
D000818:Animals; D001663:Bilirubin / Q000378:metabolism*; D019556:COS Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010766:Phosphorylation; D013379:Substrate Specificity
D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D001663:Bilirubin