Specific Nuclear Localizing Sequence Directs Two Myosin Isoforms to the Cell Nucleus in Calmodulin-Sensitive Manner

Abstract

BackgroundNuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the “cytoplasmic” myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus.Methodology/Principal FindingsWe investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1.Conclusions/SignificanceWe have shown that the novel specific NLS brings to the cell nucleus not only the “nuclear” isoform of myosin I (NM1 protein) but also its “cytoplasmic” isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

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“…We also analysed a construct that contained part of IQ domain 3 and the complete tail domain (D1-753). This construct did not localize to the nucleus at all, confirming that nuclear localization is facilitated by the recently identified NLS that is located within IQ domains 2 and 3 [19].…”

“…2A; isoform A D753-1028; isoform B D753-1028; isoform C D753-1028). However, even though all three constructs contain the IQ domain part with the recently identified NLS [19], none of these constructs showed a substantial nuclear localization (Fig. 2B).…”

“…The neck region contains three confirmed IQ domains to which regulatory calmodulin light chains bind [28]. This region also contains the recently identified NLS [19]. The C-terminal tail domain is assumed to facilitate interaction with specific binding partners [27,28].…”

“…In this context, the previously reported calmodulin-sensitive nuclear translocation of myosin IC might be of interest [19]. Calmodulin is the regulatory light chain of myosin IC and calmodulin binding to the neck is one of the mechanisms that could lead to structural changes in the above mentioned hinge region between the head and neck domain that might alter accessibility of NoLS H .…”

“…However, a recent study identified the nuclear localization signal (NLS) of myosin IC within the neck domain of the protein, and thus in a region that is common among the myosin IC isoforms [19]. Furthermore, we recently showed that the MYOIC gene encodes not two, but three isoforms (myosin IC isoforms A, B, and C; [20]).…”