An EB1-Binding Motif Acts as a Microtubule Tip Localization Signal

Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.

[1]  Nathan A. Baker,et al.  Electrostatics of nanosystems: Application to microtubules and the ribosome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Wolfgang Jahnke,et al.  Molecular basis of coiled-coil formation , 2007, Proceedings of the National Academy of Sciences.

[3]  S. Tsukita,et al.  The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules , 2000, Current Biology.

[4]  J. Cavanagh.  Protein NMR Spectroscopy: Principles and Practice , 1995 .

[5]  Anna Akhmanova,et al.  Capturing protein tails by CAP-Gly domains. , 2008, Trends in biochemical sciences.

[6]  K. Kaibuchi,et al.  Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition , 2007, Proceedings of the National Academy of Sciences.

[7]  L. Su,et al.  Characterization of Functional Domains of Human EB1 Family Proteins* , 2003, Journal of Biological Chemistry.

[8]  G. Wagner,et al.  Relaxation-Rate Measurements for 15N−1H Groups with Pulsed-Field Gradients and Preservation of Coherence Pathways , 1994 .

[9]  R. Keller,et al.  The Computer Aided Resonance Assignment Tutorial , 2004 .

[10]  G. Danuser,et al.  GSK3β phosphorylation modulates CLASP–microtubule association and lamella microtubule attachment , 2009, The Journal of cell biology.

[11]  N. Galjart,et al.  A plus-end raft to control microtubule dynamics and function. , 2003, Current opinion in cell biology.

[12]  N. Galjart,et al.  EB1 and EB3 control CLIP dissociation from the ends of growing microtubules. , 2005, Molecular biology of the cell.

[13]  W. Jahnke,et al.  Thermodynamics of the Op18/Stathmin-Tubulin Interaction* , 2003, Journal of Biological Chemistry.

[14]  M. Steinmetz,et al.  Key interaction modes of dynamic +TIP networks. , 2006, Molecular cell.

[15]  J. Shabanowitz,et al.  Aurora B Phosphorylates Centromeric MCAK and Regulates Its Localization and Microtubule Depolymerization Activity , 2004, Current Biology.

[16]  Anna Akhmanova,et al.  Structure-function relationship of CAP-Gly domains , 2007, Nature Structural &Molecular Biology.

[17]  Gary G. Borisy,et al.  Mammalian end binding proteins control persistent microtubule growth , 2009, The Journal of cell biology.

[18]  Liedewij Laan,et al.  Reconstitution of a microtubule plus-end tracking system in vitro , 2007, Nature.

[19]  G. Wider,et al.  Measuring protein concentrations by NMR spectroscopy. , 2006, Journal of the American Chemical Society.

[20]  C. Hoogenraad,et al.  Dynamic Microtubules Regulate Dendritic Spine Morphology and Synaptic Plasticity , 2009, Neuron.

[21]  David Pellman,et al.  Surfing on microtubule ends. , 2003, Trends in cell biology.

[22]  Jack Greenblatt,et al.  Methods for Measurement of Intermolecular NOEs by Multinuclear NMR Spectroscopy: Application to a Bacteriophage λ N-Peptide/boxB RNA Complex , 1997 .

[23]  G. von Dassow,et al.  MCAK associates with the tips of polymerizing microtubules , 2005, The Journal of cell biology.

[24]  Y. Goldman,et al.  Microtubule plus-end tracking by CLIP-170 requires EB1 , 2009, Proceedings of the National Academy of Sciences.

[25]  Ronald D. Vale,et al.  Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end , 2005, The Journal of cell biology.

[26]  T. Gibson,et al.  Systematic Discovery of New Recognition Peptides Mediating Protein Interaction Networks , 2005, PLoS biology.

[27]  J. Swedlow,et al.  Aurora B regulates MCAK at the mitotic centromere. , 2004, Developmental cell.

[28]  David Pellman,et al.  Microtubule “Plus-End-Tracking Proteins” The End Is Just the Beginning , 2001, Cell.

[29]  Anna Akhmanova,et al.  Tracking the ends: a dynamic protein network controls the fate of microtubule tips , 2008, Nature Reviews Molecular Cell Biology.

[30]  S. Kandels-Lewis,et al.  CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites , 2008, The Journal of cell biology.

[31]  Niels Galjart,et al.  CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex , 2005, The Journal of cell biology.

[32]  C. Hoogenraad,et al.  STIM1 Is a MT-Plus-End-Tracking Protein Involved in Remodeling of the ER , 2008, Current Biology.

[33]  I. Arnal,et al.  EB1 regulates microtubule dynamics and tubulin sheet closure in vitro , 2008, Nature Cell Biology.

[34]  M. Chen,et al.  EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration , 2004, Nature Cell Biology.

[35]  P. Tittmann,et al.  The Schizosaccharomyces pombe EB1 Homolog Mal3p Binds and Stabilizes the Microtubule Lattice Seam , 2006, Cell.

[36]  K. E. Busch,et al.  The Microtubule Plus End-Tracking Proteins mal3p and tip1p Cooperate for Cell-End Targeting of Interphase Microtubules , 2004, Current Biology.

[37]  Inke S Näthke,et al.  The adenomatous polyposis coli protein: the Achilles heel of the gut epithelium. , 2004, Annual review of cell and developmental biology.