Abstract
FEZ1 was initially described as a neuronal protein that influences axonal development and cell polarization. CLASP2 and NEK1 proteins are present in a centrosomal complex and participate in cell cycle and cell division mechanisms, but their functions were always described individually. Here, we report that NEK1 and CLASP2 colocalize with FEZ1 in a perinuclear region in mammalian cells, and observed that coiled-coil interactions occur between FEZ1/CLASP2 and FEZ1/NEK1 in vitro. These three proteins colocalize and interact with endogenous γ-tubulin. Furthermore, we found that CLASP2 is phosphorylated and interacts with active PKC isoforms, and that FEZ1/CLASP2 colocalization is inhibited by PMA treatment. Our results provide evidence that these three proteins cooperate in centrosomal functions and open new directions for future studies.
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Acknowledgements
Financially supported by: Fundação de Amparo à Pesquisa do Estado São Paulo, Conselho Nacional de Pesquisa e Desenvolvimento and LNLS. We thank Maria Eugenia Camargo and Jéssica Santana Bernachi for technical assistance. We are also grateful to Dr. Alexandre J. C. Quaresma for help in providing critical reagents.
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11010_2009_317_MOESM1_ESM.tif
Supplementary Fig. 1: The coexpression of GST-NEK1(441–621) or GST-NEK1(497–555) with 6xHis-FEZ1(229–269) results in the elution of a stably interacting complex. 6xHis FEZ1(229–269) and GST only (control), GST-NEK1(441–621) or GST-NEK1(497–555) were produced simultaneously after cotransformation of E. coli BL21 (DE3). The copurifications were performed using a GST-Trap column in an ÄKTATM FPLCTM (GE Healthcare). After mechanical lysis with a French Press, soluble and insoluble fractions were separated by centrifugation and the cleared supernatant was then loaded onto a GST-Trap column pre-equilibrated with lysis buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, and 1 mM phenylmethylsulfonyl fluoride), followed by extensive wash of the column with the same buffer. Bound proteins were eluted in a gradient of 0–100% of elution buffer (50 mM Tris–HCl and 20 mM reduced gluthatione, pH 8.0). GST only does not copurify with coexpressed 6xHis-FEZ1(229–269). The coexpressions and copurifications are shown by Coomassie stained SDS–PAGE. White arrow heads indicate GST or GST-NEK1 fusion proteins as indicated on top of the figure. Black arrow heads indicate the copurified 6xHis-FEZ1(229–269). St: molecular weight standard proteins. (TIF 1253 kb)
11010_2009_317_MOESM2_ESM.tif
Supplementary Fig. 2: Helical wheel representations of the FEZ1 interaction with NEK1 and CLASP2 coiled-coils. (A, B) Helical wheel representations of predicted hetero-dimeric coiled-coils between FEZ1 (amino acids 229–269) and NEK1 (amino acids 500–555) or CLASP2 (amino acids 1269–1303) heptad repeats. The view is from the N-terminus down to the C-terminus. (TIF 1467 kb)
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Lanza, D.C.F., Meirelles, G.V., Alborghetti, M.R. et al. FEZ1 interacts with CLASP2 and NEK1 through coiled-coil regions and their cellular colocalization suggests centrosomal functions and regulation by PKC. Mol Cell Biochem 338, 35–45 (2010). https://doi.org/10.1007/s11010-009-0317-9
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DOI: https://doi.org/10.1007/s11010-009-0317-9