Shackleford GM et al. (2001), Cloning, expression and nuclear localization of...

XB-ART-8042

BMC Genomics 2001 Jan 01;2:8. doi: 10.1186/1471-2164-2-8.

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Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones.

Shackleford GM , Ganguly A , MacArthur CA .

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BACKGROUND: Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3. RESULTS: Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus. CONCLUSIONS: Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus.

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Species referenced: Xenopus
Genes referenced: acta2 actb actl6a bcl2l11 calm2 csnk1g2 fgf8 gsk3b gys1 nasp npm1 npm3

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	Figure 1. NPM3 genomic structure and linkage to FGF8. (A) Genomic structure of NPM3. Boxed areas represent NPM3 exons. The yellow and black shadings represent translated and untranslated regions, respectively. (B) Linkage of NPM3 to FGF8. The location of genes and restriction map were determined by a combination of sequencing and restriction mapping of lambda clones and subclones in this study and a previous study [[22]]. An NPM3 cDNA probe will hybridize to restriction fragments of the following sizes in human genomic DNA: Avr II, 0.4 and 3.9 kb; Bam H I, 1.6 and 6.0 kb; Eco R V, 7.2 kb; Hind III, 6.8 kb; Kpn I, 6.6 kb; Sac I, 1.6 and 4.0 kb; Sma I, 1.0 and 5.1 kb; Stu I, 2.6 and 14.1 kb;Xho I, 8.4 kb. Restriction enzyme abbreviations: A, Avr II; B, Bam H I; H, Hind III; K, Kpn I; RV, Eco R V; N, Not I; S, Sac I; Sm, Sma I; St, Stu I; Xh, Xho I.
	Figure 2. Comparison and features of human NPM3 and mouse Npm3 amino acid sequences. Identical amino acids are denoted by an asterisk, highly similar residues by a colon and less similar residues by a period, as determined by CLUSTAL W software. The putative nuclear localization signal is overlined. Potential phosphorylation sites in NPM3 for the indicated kinases are shown, as predicted by NetPhos 2.0. Arrows denote splice points in the mRNA. Dashes represent gaps in the alignment. Kinase abbreviations: CKI, casein kinase I; CKII, casein kinase II; CaMII, calmodulin-dependent protein kinase II; GSK3, glycogen synthase kinase 3; PKA, protein kinase A; PKC, protein kinase C.
	Figure 3. A comparison of nucleophosmin/nucleoplasmin family members from metazoans. The alignment was made with CLUSTAL W. Aligned residues that were identical or similar in at least 50% of the sequences, excluding gaps, were shaded with a black or gray background, respectively; additionally, any residues that were similar to a block of identical residues were shaded in gray. Similar amino acids were grouped as follows: I, L, M, V; F, W, Y; H, K, R; D, E; N, Q; A, G; S, T; P; C. The names, species, accession numbers and references for these sequences are presented in Table 2.
	Figure 4. Dendrogram of nucleophosmin/nucleoplasmin family members. Amino acid sequences from the 16 proteins shown were aligned with CLUSTAL W, and a dendrogram was produced using the TreeTop algorithm as described in Materials and Methods. The 16 sequences included 14 from the nucleophosmin/nucleoplasmin family (see Table 2 and Figure 3) and two other histone-binding proteins (NASP and N1/N2), which are closely related to each other but unrelated to the nucleophosmin/nucleoplasmin family. Bootstrap significance values are shown at the branchpoints for 100 resamplings. The analysis suggests that NPM3 is more related to nucleoplasmins than to nucleophosmins and that NPM3 is closely related to Xenopus NO29.
	Figure 5. Northern blot expression analysis of NPM3 in human tissues. Upper panels, hybridization with the human NPM3 cDNA probe. Lower panels, hybridization with the mouse beta-actin cDNA probe. The location of the molecular weight standards in kb is indicated to the left of the blots.
	Figure 6. Immunoblot analysis of the cellular localization of HA-tagged NPM3. Protein samples (50 mg) were subjected to SDS-PAGE, electrophoretic transfer to nitrocellulose and immunoblotting with an anti-HA antibody. Media, cytoplasm and nucleus are the subcellular fractions analyzed. The locations of the molecular weight standards in kDa are indicated to the left of the blot. Arrows indicate bands associated with HA-tagged NPM3. (A) cells mock-transfected; (B) cells transfected with antisense HA-tagged NPM3 cDNA construct; (C) cells transfected with sense HA-tagged NPM3 cDNA construct.

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