Inhibition of chloroplast protein phosphorylation by cAMP in Lemna paucicostata 6746

Abstract

Phosphorylation was carried out in chloroplasts, purified on sucrose density gradient, using [γ-³²P] ATP. Among several phosphopolypeptides detected, polypeptides in the M_r range of 24-22 k, which are known to constitute the light-harvesting chlorophyll a/b-binding protein complex (LHCP), and 16-18 k were highly phosphorylated. Cyclic AMP (100 μM) decreased the overall phosphorylation of chloroplast polypeptides and its effect was more striking on LHCP. Other nucleotides such as cGMP, 2',3'-cAMP, 2',3'-cGMP and 5'-AMP, at equimolar level, did not show any significant effect on phosphorylation of chloroplast polypeptides. The effect of cAMP was also studied on light-dependent in vitro phosphorylation of chloroplast polypeptides by incubating a crude preparation of intact chloroplasts with [³²Pi]. In dark control, two polypeptides of M_r 66 and 64 k were distinctly phosphorylated. On illumination, several other polypeptides in the range of 97-12 k were also phosphorylated, with the 26-24 k LHCP fraction being the major phosphopolypeptides. Cyclic AMP (at 100 μM and 1 mM) specifically caused a decrease in phosphorylation of the 26-24, 21-20 and 12 k polypeptides. The phosphorylation status of the 66-64 k doublet, as attained in dark, remained unaffected in the presence of light and/or cAMP. Sodium fluoride, a protein phosphatase inhibitor, enhanced the phosphorylation of 26-24, 21-20 and 12 k polypeptides but did not affect the inhibitory action of cAMP. It is thus likely that cAMP down-regulates protein kinase(s) responsible for the phosphorylation of these polypeptides. The present study also provides evidence that this protein kinase is sensitive to staurosporine, a protein kinase inhibitor.