Clock and Bmal1: The Positive Arm Proteins of Circadian Rhythm are not Regulated by Tyrosine Phosphorylation in Morphine Dependence

The present study has been designed to determine any role of tyrosine phosphorylation in regulating clock components during morphine dependence. The involvement of clock components in opioid addiction is strongly approved by a number of observations which showed that circadian rhythmicity can regulate different aspects of addiction such as drug-seeking, craving, drug reward and reinforcement. These clock molecules at their protein levels are regulated by different post-translational modifications and among them phosphorylation is the most significant one which regulates their localization, functions and stability. In this context, unlike tyrosine phosphorylation the role of serine/ threonine phosphorylation of clock components is well studied. However, sequence analyses of clock members have shown number of potential tyrosine phosphorylation sites. For our work, we have selected Clock and Bmal1 proteins which are the members of positive arm of circadian rhythm and their hetero-dimeric form regulates the transcription of negative arm proteins Per and Cry. At first, animals were divided into two groups: control/saline treated and morphine treated. Chronic morphine treatment-induced dependence in mice was determined by examining the naloxone precipitated withdrawal syndromes. Then we determine the tyrosine phosphorylation profile of whole proteins present in cortex or hippocampus or striatum of the mice brains. A significant increase of tyrosine phosphorylation was observed in striatum. Although, immunoprecipitation of the striatum tissue did not find tyrosine phosphorylated Clock or Bmal1 proteins. Even treatment of cultured cells with H₂O₂, which caused global tyrosine phosphorylation, was unable to induce tyrosine phosphorylation of Clock and Bmal1. Our studies showed that both in morphine dependence and H₂O₂ induced global tyrosine phosphorylation conditions, Clock and Bmal1 proteins were not become phosphorylated at tyrosine residues despite having number of potential tyrosine phosphorylation sites.