Nuclear variants of bone morphogenetic proteins

Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah, USA.
BMC Cell Biology (Impact Factor: 2.34). 03/2010; 11(1):20. DOI: 10.1186/1471-2121-11-20
Source: PubMed


Bone morphogenetic proteins (BMPs) contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts.
In all three proteins, a bipartite nuclear localization signal (NLS) was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5) containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle.
The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.

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    • "48 hrs after transfection, cells were fixed using 4% paraformaldehyde, nuclei were stained with a 1 : 1000 dilution of TOPRO-3 iodide (Invitrogen Corporation, Carlsbad, CA), and slides were mounted in Fluoromount-G (Southern Biotech, Birmingham , AL) and coverslipped. Cells were imaged and nuclear localization was quantified using an Olympus IX81 laser confocal microscope as previously described [20]. To assess BMP2 secretion, RCS cells were seeded in 25 cm 2 culture flasks and transfected with HA-tagged expression plasmids wtBmp2/HA or RKRmBmp2/HA (both containing the HA tag at the C-terminus of BMP2) as described above. "
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    ABSTRACT: We recently reported a novel form of BMP2, designated nBMP2, which is translated from an alternative downstream start codon and is localized to the nucleus rather than secreted from the cell. To examine the function of nBMP2 in the nucleus, we engineered a gene-targeted mutant mouse model (nBmp2NLS(tm)) in which nBMP2 cannot be translocated to the nucleus. Immunohistochemistry demonstrated the presence of nBMP2 staining in the myonuclei of wild type but not mutant skeletal muscle. The nBmp2NLS(tm) mouse exhibits altered function of skeletal muscle as demonstrated by a significant increase in the time required for relaxation following a stimulated twitch contraction. Force frequency analysis showed elevated force production in mutant muscles compared to controls from 10 to 60 Hz stimulation frequency, consistent with the mutant muscle's reduced ability to relax between rapidly stimulated contractions. Muscle relaxation after contraction is mediated by the active transport of Ca(2+) from the cytoplasm to the sarcoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA), and enzyme activity assays revealed that SERCA activity in skeletal muscle from nBmp2NLS(tm) mice was reduced to approximately 80% of wild type. These results suggest that nBMP2 plays a role in the establishment or maintenance of intracellular Ca(2+) transport pathways in skeletal muscle.
    11/2013; 2013(698):125492. DOI:10.1155/2013/125492
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    • "Although GDF5 is a secreted protein, we also observed specific GDF5 immunoreactivity in pyramidal cell nuclei. Nuclear GDF5 has been reported in cell lines, and GDF5, BMP2 and BMP4 possess a bipartite nuclear localisation signal that overlaps the cleavage site of the respective proproteins (Felin et al., 2010). Gdf5, Bmp2 and Bmp4 mRNAs are initiated from alternative start codons to generate proteins that either possess an N-terminal signal peptide and are secreted or lack a signal peptide, retain an intact nuclear localisation signal and are translocated to the nucleus (Felin et al., 2010). "
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    ABSTRACT: Dendrite size and morphology are key determinants of the functional properties of neurons. Here, we show that growth differentiation factor 5 (GDF5), a member of the bone morphogenetic protein (BMP) subclass of the transforming growth factor β superfamily with a well-characterised role in limb morphogenesis, is a key regulator of the growth and elaboration of pyramidal cell dendrites in the developing hippocampus. Pyramidal cells co-express GDF5 and its preferred receptors, BMP receptor 1B and BMP receptor 2, during development. In culture, GDF5 substantially increased dendrite, but not axon, elongation from these neurons by a mechanism that depends on activation of SMADs 1/5/8 and upregulation of the transcription factor HES5. In vivo, the apical and basal dendritic arbours of pyramidal cells throughout the hippocampus were markedly stunted in both homozygous and heterozygous Gdf5 null mutants, indicating that dendrite size and complexity are exquisitely sensitive to the level of endogenous GDF5 synthesis.
    Development 10/2013; 140(23). DOI:10.1242/dev.101378 · 6.46 Impact Factor
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    ABSTRACT: In general, the output of a DDS (direct digital synthesis) system will inherently include spurious signals due to two effects: (1) phase truncation errors; and (2) phase to amplitude conversion errors. Phase truncation errors have traditionally been analyzed separately from the fundamental DDS output signal making the derivations awkward and hard to follow. We have recognized that by: (1) only dealing with the active portion of the phase accumulator; (2) carefully reformulating the instantaneous phase; and (3) including all possible initial conditions, the spectral analyses of the DDS output signal can be performed directly, exactly and in a way that is much easier to follow. Our analysis approach results in a simple formulation of the frequency locations and spacing of the phase truncation spurs. We have also recognized that, as long as the phase-to-amplitude conversion errors are "static" in nature, all possible DDS output spectra are inter-related. This is true without any a priori knowledge of the errors. The equations for this interrelationship have been derived in this paper. They show that if a specific DDS spectrum is known, all other possible spectra can be derived from it. We referred to this specific spectrum as the mother spectrum. Also from the properties of these inter-relationships, a different insight has been given to the interpretation of the phase truncation spurs. Based on our derivations, it has also been shown that observations of the DDS spectrum may be affected by using a phase-locked loop phase detector.
    Frequency Control Symposium and Exposition, 2004. Proceedings of the 2004 IEEE International; 09/2004
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