Dual nuclear import mechanisms of sex determining factor SRY: intracellular Ca2+ as a switch

The sex-determining region on the Y chromosome (SRY) has 2 nuclear localization signals (NLSs) that flank the DNA binding high mobility group (HMG) domain; the beta-NLS and the CaM-NLS, which mediate nuclear transport through importin beta 1 (Imp beta 1) and the calcium-binding protein calmodulin (CaM), respectively. Here we reconstitute the nuclear import mediated by the 2 NLSs for the first time in vitro, establishing Ran independence of CaM-NLS-dependent transport. The beta- and CaM-NLSs were found to be independently functional out of the context of the SRY HMG domain, dependent on Imp beta 1 and CaM binding, respectively. Intriguingly, direct protein binding assays also indicated competitive binding of Imp beta 1 and CaM to the SRY HMG domain. To assess the potential role of intracellular calcium in modulating SRY nuclear accumulation, Cos-7 cells expressing SRY and control constructs were treated with agents elevating or reducing intracellular Ca2+ levels. The in vivo results, supported by experiments in vitro where transport was assessed with or without 2 mu M Ca2+, indicate a Ca2+-dependent mode of nuclear transport via the CaM-NLS/CaM, with inhibition of beta-NLS/Imp beta 1-mediated nuclear import by intracellular Ca2+. The results imply mutual exclusivity of nuclear transport via the 2 NLSs with intracellular Ca2+ as the switch between the 2.-Kaur, G. Jans, D. A. Dual nuclear import mechanisms of sex determining factor SRY: intracellular Ca2+ as a switch. FASEB J. 25, 665-675 (2011). www.fasebj.org