Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal

The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins. Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids. Two putative NLSs were identified in PLAG1. The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1. Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import. The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1. These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site.     

Isolation of nlz2 and characterization of essential domains in Nlz family proteins

In this study, we first cloned nlz2, a second zebrafish member of the nlz-related zinc-finger gene family. nlz2 was expressed together with nlz1 in a broad posterior domain during gastrula stages as well as at the midbrain-hindbrain boundary and in the hindbrain caudal to rhombomere 4 during segmentation. nlz2 was also expressed in regions distinct from nlz1, notably in the forebrain, midbrain, and trunk. Misexpression of nlz2 in zebrafish embryos disrupted gene expression in the rostral hindbrain, similar to the effect of misexpressing nlz1. We next compared the nlz1 and nlz2 sequences to identify and characterize domains conserved within this family. We found a C-terminal domain required for nuclear localization and two conserved domains (the Sp motif and a putative C(2)H(2) zinc finger) required for nlz1 function. We also demonstrate that Nlz1 self-associated via its C terminus, interacted with Nlz2, and bound to histone deacetylases. Last, we found two forms of Nlz1 generated from alternative translation initiation sites in vivo. These forms have distinct activities, apparently depending on the function of the N-terminal Sp motif. Our data demonstrate that nlz2 functions similarly to nlz1 and define conserved domains essential for nuclear localization, self-association, and corepressor binding in this novel family of zinc-finger genes.     

RNF151, a testis-specific RING finger protein, interacts with dysbindin

RING finger proteins play important roles in spermatogenesis. Here, we report that a novel RING finger protein RNF151, with a C3HC4-type RING finger domain, a putative nuclear localization signal (NLS), and a TRAF-type zinc finger domain, was exclusively expressed in the mouse testis and developmentally regulated during spermatogenesis. While RNF151 mRNA was present in round spermatids, its protein was expressed in elongating spermatids of the stage VIII-IX seminiferous tubules. The NLS together with the RING domain were necessary and sufficient for the nuclear localization of RNF151-EGFP in transfected cells. Yeast two-hybrid screening identified the physical interaction of mouse RNF151 and dysbindin, which was confirmed by the co-immunoprecipitation of the proteins and by their co-localization in intact cells. As dysbindin has lately been shown to be involved in membrane biogenesis and fusion, a key process for acrosome formation, we propose that RNF151 may play a role in acrosome formation.