Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation

J Biol Chem. 2001 Apr 6;276(14):10929-34. doi: 10.1074/jbc.M010753200. Epub 2001 Jan 17.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bilirubin / metabolism*
  • COS Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Oxidoreductases / metabolism*
  • Oxidoreductases Acting on CH-CH Group Donors*
  • Phosphorylation
  • Substrate Specificity

Substances

  • Oxidoreductases
  • Oxidoreductases Acting on CH-CH Group Donors
  • biliverdin reductase
  • Bilirubin