Nucleolar localization of SmMAK16 protein from Schistosoma mansoni is regulated by three distinct signals that function independent of pH or phosphorylation status

Abstract

SmMAK16 from the trematode Schistosoma mansoni is a protein that is known to localize in the nucleolus. Recent findings show that SmMAK16 is involved in 60S ribosomal subunit synthesis. Although the SmMAK16 protein contains putative nuclear localization signals (NLS), little is known about their precise function, redundancy or regulation. The goal of the current study was to identify and characterize the presence and functional regulation of the localization signals in SmMAK16. The SmMAK16 coding sequence and specific fragments were individually cloned in-frame into the pEGFP-C2 expression vector to encode Green Fluorescent Protein (GFP) fusion proteins. Constructs were individually transfected into COS-7 cells and fluorescent microscopy used to determine the cellular location and thus the presence of signals regulating nuclear and nucleolar localization. SmMAK16 was found to contain two NLSs and one nucleolar localization signal (NoLS). One of the signals contains a sequence identical to an established nucleolar detention signal that reportedly functions only under acidic cellular conditions. The localization of the SmMAK16-GFP constructs was analyzed under acidic conditions; however, altering pH did not influence the localization of SmMAK16. It has been previously reported that casein kinase 2 (CK2) can phosphorylate SmMAK16 at serines adjacent to one of the NLSs. One of these CK2 sites and the adjacent NLS are conserved with that of the SV40 Large T Antigen (LTA) and phosphorylation of this site in the SV40 LTA regulates the kinetics of the NLS. To discover if kinetic regulation also occurs in SmMAK16, mutant and wild type SmMAK16-GFP proteins were purified and injected into individual COS-7 cells. No difference in the rate of transport was found between wt and mutant SmMAK16 proteins. Therefore, SmMAK16 localizes to the nucleolus using three separate signals, two NLSs and one NoLS, however, these signals appear to function independently of pH and phosphorylation by CK2.