Nuclear-to-cytoplasmic Relocalization of the Proliferating Cell Nuclear Antigen (PCNA) during Differentiation Involves a Chromosome Region Maintenance 1 (CRM1)-dependent Export and Is a Prerequisite for PCNA Antiapoptotic Activity in Mature Neutrophils

Abstract

Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells, is a key regulator of neutrophil survival. In neutrophils, PCNA was localized exclusively in the cytoplasm due to its nuclear-to-cytoplasmic relocalization during granulocytic differentiation. We showed here that leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1) exportin, inhibited PCNA relocalization during granulocytic differentiation of HL-60 and NB4 promyelocytic cell lines and of human CD34(+) primary cells. Using enhanced green fluorescent protein fusion constructs, we have demonstrated that PCNA relocalization involved a nuclear export signal (NES) located from Ile-11 to Ile-23 in the PCNA sequence. However, this NES, located at the inner face of the PCNA trimer, was not functional in wild-type PCNA, but instead, was fully active and leptomycin B-sensitive in the monomeric PCNAY114A mutant. To test whether a defect in PCNA cytoplasmic relocalization would affect its antiapoptotic activity in mature neutrophils, a chimeric PCNA fused with the SV40 nuclear localization sequence (NLS) was generated to preclude its cytoplasmic localization. As expected, neutrophil-differentiated PLB985 cells expressing ectopic SV40NLS-PCNA had an increased nuclear PCNA as compared with cells expressing wild-type PCNA. Accordingly, the nuclear PCNA mutant did not show any antiapoptotic activity as compared with wild-type PCNA. Nuclear-to-cytoplasmic relocalization that occurred during myeloid differentiation is essential for PCNA antiapoptotic activity in mature neutrophils and is dependent on the newly identified monomerization-dependent PCNA NES.

Proliferating cell nuclear antigen in neutrophil fate

“…To ascertain whether the anti‐apoptotic function of PCNA in mature neutrophils is due to this cytosolic localization, we studied the function of a PCNA mutant fused with the nuclear localization sequence (NLS) of the SV40 virus to direct PCNA to the nucleus and preclude its cytosolic localization of PCNA. As expected and in contrast to wildtype PCNA, the nuclear SV40NLS‐PCNA mutant did not provide any anti‐apoptotic activity when ectopically expressed in DMF‐differentiated PLB985 cells . Cytosolic relocalization of PCNA seems to be part of the final differentiation of granulocytes involved in the survival mechanisms of these cells.…”

“…Indeed, by fusing this sequence to the EGFP protein either at its amino‐ or carboxy‐terminal portion, we observe the nuclear export of the chimeric EGFP in a CRM1‐dependent manner as the process is inhibited by leptomycin B. As expected, point mutations at the critical lysine residues within the sequence totally abolish this export . However, because this NES sequence is located at the inner face of the PCNA trimer, this NES is not functional within trimeric PCNA and NES mutation within the PCNA sequence does not result in EGFP nuclear accumulation.…”

“…Pertinently, in the early stages of our understanding the dogma was that PCNA localization and functions were exclusively restricted to the nuclear compartment. Contrary to this, we discovered that in mature neutrophils, PCNA is present exclusively within the cytosol and our subsequent identification of a functional nuclear export sequence (NES) in PCNA corroborated the fact that it also exerts important function outside of the nucleus . As neutrophils are highly differentiated and non‐proliferating cells, they constituted a unique model to decipher the cell cycle‐independent functions of PCNA.…”

“…In CD34 + cells, PCNA is detectable exclusively within the nucleus whereas at the end of the differentiation process, in cells with neutrophil‐like nuclei, PCNA is located exclusively within the cytoplasm, similar to what was observed in mature peripheral neutrophils. The first evidence of an active nuclear export was provided by the effect of leptomycin B, an inhibitor of the Crm1 exportin, which inhibited the nuclear‐to‐cytoplasmic relocalization occurring at the end of the differentiation process . Similar nuclear‐to‐cytoplasmic relocalization of PCNA is observed using ATRA‐induced differentiation in both NB4 and HL60 cell lines.…”

Classification of protein motifs based on subcellular localization uncovers evolutionary relationships at both sequence and functional levels

“…Cells are highly compartmentalized, limiting most proteins to specific subcellular regions or organelles. The distribution of a protein plays a critical role in determining its activity [1]. For example, a DNA-binding protein would not be able to bind DNA if it were restricted to the endoplasmic reticulum (ER).…”