Conserved Motif of CDK5RAP2 Mediates Its Localization to Centrosomes and the Golgi Complex*

As the primary microtubule-organizing centers, centrosomes require γ-tubulin for microtubule nucleation and organization. Located in close vicinity to centrosomes, the Golgi complex is another microtubule-organizing organelle in interphase cells. CDK5RAP2 is a γ-tubulin complex-binding protein and functions in γ-tubulin attachment to centrosomes. In this study, we find that CDK5RAP2 localizes to the Golgi complex in an ATP- and centrosome-dependent manner and associates with Golgi membranes independently of microtubules. CDK5RAP2 contains a centrosome-targeting domain with its core region highly homologous to the Motif 2 (CM2) of centrosomin, a functionally related protein in Drosophila. This sequence, referred to as the CM2-like motif, is also conserved in related proteins in chicken and zebrafish. Therefore, CDK5RAP2 may undertake a conserved mechanism for centrosomal localization. Using a mutational approach, we demonstrate that the CM2-like motif plays a crucial role in the centrosomal and Golgi localization of CDK5RAP2. Furthermore, the CM2-like motif is essential for the association of the centrosome-targeting domain to pericentrin and AKAP450. The binding with pericentrin is required for the centrosomal and Golgi localization of CDK5RAP2, whereas the binding with AKAP450 is required for the Golgi localization. Although the CM2-like motif possesses the activity of Ca2+-independent calmodulin binding, binding of calmodulin to this sequence is dispensable for centrosomal and Golgi association. Altogether, CDK5RAP2 may represent a novel mechanism for centrosomal and Golgi localization.

[1]  Yi Xiang,et al.  ERK regulates Golgi and centrosome orientation towards the leading edge through GRASP65 , 2008, The Journal of cell biology.

[2]  F. Perez,et al.  The Golgi complex is a microtubule-organizing organelle. , 2001, Molecular biology of the cell.

[3]  C. Rieder,et al.  The Sudden Recruitment of γ-Tubulin to the Centrosome at the Onset of Mitosis and Its Dynamic Exchange Throughout the Cell Cycle, Do Not Require Microtubules , 1999, The Journal of cell biology.

[4]  B. Delaval,et al.  Pericentrin in cellular function and disease , 2010, The Journal of cell biology.

[5]  J. Salisbury.  Centrosomes: Coiled-Coils Organize the Cell Center , 2003, Current Biology.

[6]  C. Vidair,et al.  Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome , 1998, The Journal of cell biology.

[7]  T. Stearns,et al.  δ-Tubulin and ɛ-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function , 1999, Nature Cell Biology.

[8]  M. Bornens,et al.  Microtubule nucleation at the cis‐side of the Golgi apparatus requires AKAP450 and GM130 , 2009, The EMBO journal.

[9]  I. Sandoval,et al.  Membrane flow through the Golgi apparatus: specific disassembly of the cis-Golgi network by ATP depletion. , 1999, Journal of cell science.

[10]  D. A. Stirling,et al.  Interaction with calmodulin is required for the function of Spc110p, an essential component of the yeast spindle pole body. , 1994, The EMBO journal.

[11]  S. Jin,et al.  Myomegalin Is a Novel Protein of the Golgi/Centrosome That Interacts with a Cyclic Nucleotide Phosphodiesterase* , 2001, The Journal of Biological Chemistry.

[12]  J. Maller,et al.  A Centrosomal Localization Signal in Cyclin E Required for Cdk2-Independent S Phase Entry , 2004, Science.

[13]  D. Dransfield,et al.  AKAP350, a Multiply Spliced Protein Kinase A-anchoring Protein Associated with Centrosomes* , 1999, The Journal of Biological Chemistry.

[14]  J. Yates,et al.  Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network. , 2007, Developmental cell.

[15]  M. Larocca,et al.  AKAP350 interaction with cdc42 interacting protein 4 at the Golgi apparatus. , 2004, Molecular biology of the cell.

[16]  T. Okanoue,et al.  Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes. , 2001, Molecular biology of the cell.

[17]  M. Bornens,et al.  Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles. , 1996, Journal of cell science.

[18]  R. Qi,et al.  Interaction of CDK5RAP2 with EB1 to track growing microtubule tips and to regulate microtubule dynamics. , 2009, Molecular biology of the cell.

[19]  S. Duensing,et al.  The forkhead-associated domain protein Cep170 interacts with Polo-like kinase 1 and serves as a marker for mature centrioles. , 2005, Molecular biology of the cell.

[20]  S. Munro,et al.  The PACT domain, a conserved centrosomal targeting motif in the coiled‐coil proteins AKAP450 and pericentrin , 2000, EMBO reports.

[21]  Kyung S. Lee,et al.  Requirement of hCenexin for Proper Mitotic Functions of Polo-Like Kinase 1 at the Centrosomes , 2006, Molecular and Cellular Biology.

[22]  T. Davis,et al.  Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents , 1996, The Journal of cell biology.

[23]  J. Lippincott-Schwartz,et al.  Brefeldin A: insights into the control of membrane traffic and organelle structure , 1992, The Journal of cell biology.

[24]  M. Bornens,et al.  Dissociating the centrosomal matrix protein AKAP450 from centrioles impairs centriole duplication and cell cycle progression. , 2003, Molecular biology of the cell.

[25]  Andrew W. Folkmann,et al.  Golgi-derived CLASP-dependent Microtubules Control Golgi Organization and Polarized Trafficking in Motile Cells , 2009, Nature Cell Biology.

[26]  E. Nigg,et al.  Cep68 and Cep215 (Cdk5rap2) are required for centrosome cohesion , 2007, Journal of Cell Science.

[27]  R. Qi,et al.  Calmodulin Binding and Cdk5 Phosphorylation of p35 Regulate Its Effect on Microtubules* , 2008, Journal of Biological Chemistry.

[28]  L. Mir,et al.  Centriole Disassembly In Vivo and Its Effect on Centrosome Structure and Function in Vertebrate Cells , 1998, The Journal of cell biology.

[29]  Michel Bornens,et al.  GMAP-210 Recruits γ-Tubulin Complexes to cis-Golgi Membranes and Is Required for Golgi Ribbon Formation , 2004, Cell.

[30]  Hussain Jafri,et al.  A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size , 2005, Nature Genetics.

[31]  Marc S. Sherman,et al.  Calmodulin Target Database , 2004, Journal of Structural and Functional Genomics.

[32]  J. Maller,et al.  Calcium, Calmodulin, and CaMKII Requirement for Initiation of Centrosome Duplication in Xenopus Egg Extracts , 2002, Science.

[33]  A Khodjakov,et al.  A synergy of technologies: combining laser microsurgery with green fluorescent protein tagging. , 1997, Cell motility and the cytoskeleton.

[34]  T. Stearns,et al.  Plk1-Dependent Recruitment of γ-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components , 2009, PloS one.

[35]  T. Davis,et al.  The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae , 1993, Molecular and cellular biology.

[36]  J. B. Rattner,et al.  CDK5RAP2 is a pericentriolar protein that functions in centrosomal attachment of the gamma-tubulin ring complex. , 2008, Molecular biology of the cell.

[37]  K. Takata,et al.  Localization of Golgi 58K protein (formiminotransferase cyclodeaminase) to the centrosome , 2006, Histochemistry and Cell Biology.

[38]  Y. Ono,et al.  Characterization of a Novel Giant Scaffolding Protein, CG-NAP, That Anchors Multiple Signaling Enzymes to Centrosome and the Golgi Apparatus* , 1999, The Journal of Biological Chemistry.

[39]  J. Lippincott-Schwartz,et al.  Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. , 1996, Molecular biology of the cell.

[40]  A. Merdes,et al.  Assembly of centrosomal proteins and microtubule organization depends on PCM-1 , 2002, The Journal of cell biology.

[41]  L. Machesky,et al.  Microtubule involvement in NIH 3T3 Golgi and MTOC polarity establishment , 2003, Journal of Cell Science.

[42]  K. Sawin,et al.  Microtubule Nucleation at Non-Spindle Pole Body Microtubule-Organizing Centers Requires Fission Yeast Centrosomin-Related Protein mod20p , 2004, Current Biology.

[43]  M. Bornens,et al.  Cloning and characterization of a cDNA encoding an A‐kinase anchoring protein located in the centrosome, AKAP450 , 1999, The EMBO journal.

[44]  T. Megraw,et al.  Proper recruitment of gamma-tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires Centrosomin Motif 1. , 2007, Molecular biology of the cell.

[45]  Lan Huang,et al.  GM130-dependent control of Cdc42 activity at the Golgi regulates centrosome organization. , 2009, Molecular biology of the cell.

[46]  L. Klein-Hitpass,et al.  Centrosomal CAP350 protein stabilises microtubules associated with the Golgi complex , 2007, Journal of Cell Science.

[47]  R. Sannerud,et al.  The function of the intermediate compartment in pre-Golgi trafficking involves its stable connection with the centrosome. , 2009, Molecular biology of the cell.

[48]  T. Davis,et al.  Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe. , 2002, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[49]  E. Schiebel,et al.  The spacer protein Spc110p targets calmodulin to the central plaque of the yeast spindle pole body. , 1996, Journal of cell science.