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NEK1 has at least two functional NLSs
Fig. 3.
NEK1 has two functional nuclear localization signals. IMCD3 PLX4720 were transfected with GFP-NEK1 fusion constructs (green) using the indicated residues of NEK1, then fixed for immunofluorescence and stained with DAPI (blue). The frequency of the observed localization of each construct [nuclear (N), cytoplasmic (C), or both (N/C)] is indicated. Scale bar represents 5 μm.
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We proceeded to test functionality of the predicted bipartite NLS, which is in the coiled-coil domain, by mutating two stretches of basic amino acids in the NLS, KK357AG and KR366AG, in the 353–686 construct. As predicted, nuclear localization of this construct is severely disrupted (Fig. 3H). Including the basic domain in NLS-mutated EGFP-NEK1 (residues 258–686, Fig. 3I) rescues nuclear localization of the construct. Taken together, these data confirm that the predicted NLS is functional, and that the basic domain harbors a second cryptic NLS. Our data demonstrate that each of the two NLSs is sufficient on its own to direct nuclear localization of NEK1.