Nuclear localization signals (NLSs) endowed the dual-specificity protein phosphatase-4 (DUSP4) of Crassostrea gigas...

Dual-specificity protein phosphatase 4 (DUSP4), a critical negative regulator of the mitogen-activated protein kinase (MAPK) signaling pathway, enters the nucleus via conserved nuclear localization sequences (NLS) to dephosphorylate key kinases in vertebrates. However, the functional and evolutionary significance of these NLSs in invertebrates remains unclear. We investigated the effects of the typical DUSP4 NLS RRRAK and KKRx₁₀KQRR on the subcellular localization and phosphatase function of the CgDUSP4 protein in the Pacific oyster (Crassostrea gigas). Comparative protein sequence analysis revealed that CgDUSP4 lacks the conserved NLSs. The mutant CgDUSP4 (mCgDUSP4) with typical NLSs was generated to investigate the effects of NLS on the subcellular localization and function of CgDUSP4. Notably, although mCgDUSP4 was not completely localized to the nucleus, it endowed mCgDUSP4 with the novel ability to specifically dephosphorylate p38 and JUN N-terminal kinase (JNK). This reveals that substrate recognition and nuclear import are separable functions encoded within these motifs. These findings provide mechanistic insights into the molecular evolution of DUSP4 and contribute to understanding how structural innovations in regulatory proteins shape the diversification of signaling pathways across metazoans.