Journal des bioprocédés et des biotechniques

A Dynamic View of Protein Phosphatase 1 Interaction with its Inhibitors

Abstract

Sudhish Mishra, Mandal A, Gupta RC and Mandal PK

Protein phosphatase 1 (PP1) is well known for their role in signal transduction and protein function. Together with its inhibitors 1 and 2, it regulates wide variety of cellular activities. We have amplified catalytic subunit of PP1 (PP1c) and its inhibitors 1 (I-1) and 2 (I-2) from dog cardiac mRNA by rt-PCR and cloned into bacterial expression vector pCRT7. Cloned genes were expressed in E. coli BL21 (DE3) pLysS by IPTG induction. Functional positive clones were identified by western blotting of bacterial lysate and polymerase chain reaction. Double transformed bacterial cells were also generated by transforming PP1c clone with either I-1 or I-2. Activity of PP1 was analyzed in whole bacterial lysate by measuring dephosphorylation of phos b. Activities of inhibitors were analyzed by their capability to inhibit PP1 from dephosphorylation of phos b. Our findings indicate differential regulation of PP1 by I-1 and I-2. Both expressed recombinant inhibitors 1 and 2 have high potency for inhibition of PP1 activity. Interestingly, I-2 co-expression caused increase in PP1c expression but no change in expression was observed when co-expressed with I-1.