Regulación de la función celular de la GTPasa Gpn3 a través de la modulación de su localización subcelular en células humanas

Abstract

The best-known function of the essential GPN-loop GTPase Gpn3 is to contribute to RNA polymerase II assembly, a prerequisite for its nuclear targeting. Although this process occurs in the cytoplasm, we have previously shown that Gpn3 enters the cell nucleus before being polyubiquitinated. Here, we show that inhibiting Crm1- mediated nuclear export with leptomycin B or the proteasome with MG132 caused the nuclear accumulation of endogenous and recombinant Gpn3RFlag in MCF-12A cells. When added simultaneously, leptomycin B and MG132 had an additive effect. Analysis of Gpn3 primary sequence revealed the presence of at least five nuclear export sequence (NES) motifs, with four having a higher exposure to the solvent in the GTP-bound than GDP-bound state in a structural Gpn3 model. Inactivation of any of these NESes led to some degree of Gpn3 nuclear accumulation, although mutating NES1 or NES3 had the more robust effect. MCF-12A cells expressing exclusively NES-deficient versions of Gpn3RFlag proliferated slower than cells expressing Gpn3RFlag wt, indicating that nuclear export is important for Gpn3 cellular function. Next, we searched for physiological conditions that may regulate Gpn3 nucleocytoplasmic shuttling. Interestingly, whereas Gpn3RFlag was mostly nuclear in sparsely growing MCF-12A cells, it was exclusively cytoplasmic in confluent cells. Furthermore, Gpn3RFlag was cytoplasmic, mostly perinuclear, in sparse but starved MCF-12A cells, and serum-stimulation caused a rapid, although transient, Gpn3RFlag nuclear accumulation. We conclude that Gpn3 nucleocytoplasmic shuttling is regulated by cell density and growth factors, and propose that Gpn3 has an unknown nuclear function positively linked to cell growth and/or proliferation.