Inhibition of Protein 4.1 R and NuMA Interaction by Mutagenization of Their Binding‐Sites Abrogates Nuclear Localization of 4.1 R

Protein 4.1R(4.1R) is a multifunctional structural protein recently implicated in nuclear assembly and cell division. We earlier demonstrated that 4.1R forms a multiprotein complex with mitotic spindle and spindle pole organizing proteins, such as NuMA, dynein, and dynactin, by binding to residues 1788–1810 of NuMA through amino acids encoded by exons 20 and 21 in 24 kD domain. Employing random‐and site‐directed mutagenesis combined with glycine‐ and alanine‐scanning, we have identified amino acids of 4.1 R and NuMA that sustain their interaction, and have analyzed the effect of mutating the binding sites on their intracellular colocalization. We found that V762, V765, and V767 of 4.1 R, and 11800, 11801,11803, Tl 804, and M1805 of NuMA are necessary for their interaction. GST‐fusion peptides of the 4.1R24 kD domain bound to residues 1785–2115 of NuMA in in vitro binding assays, but the binding was inhibited by alanine substitutions of V762, V765, and V767 of 4.1 R, or residues 1800–1805 of NuMA. Additionally, expression of variants of 4.1 R or NuMA that inhibit their in vitro binding also abrogated nuclear localization of 4.1 Rand colocalization with NuMA. Our findings suggest a crucial role of 4.1 R/NuMA interaction in localization and function of 4.1 R in the nucleus.

[1]  J. Spence,et al.  Downregulation of Protein 4.1R, a Mature Centriole Protein, Disrupts Centrosomes, Alters Cell Cycle Progression, and Perturbs Mitotic Spindles and Anaphase , 2008, Molecular and Cellular Biology.

[2]  E. Benz,et al.  Protein 4.1R, a Microtubule-associated Protein Involved in Microtubule Aster Assembly in Mammalian Mitotic Extract* , 2004, Journal of Biological Chemistry.

[3]  R. Heald,et al.  Two Protein 4.1 Domains Essential for Mitotic Spindle and Aster Microtubule Dynamics and Organization in Vitro* , 2004, Journal of Biological Chemistry.

[4]  J. Conboy,et al.  Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct N-termini. , 2003, Blood.

[5]  J. A. Gimm,et al.  Two Distinct Domains of Protein 4.1 Critical for Assembly of Functional Nuclei in Vitro * , 2002, The Journal of Biological Chemistry.

[6]  F. Delhommeau,et al.  A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells. , 2002, Blood.

[7]  I. Correas,et al.  4.1R Proteins Associate with Interphase Microtubules in Human T Cells , 2001, The Journal of Biological Chemistry.

[8]  T. Tang,et al.  Protein 4.1 R-135 Interacts with a Novel Centrosomal Protein (CPAP) Which Is Associated with the γ-Tubulin Complex , 2000, Molecular and Cellular Biology.

[9]  C. Tang,et al.  Protein 4.1R binding to eIF3-p44 suggests an interaction between the cytoskeletal network and the translation apparatus. , 2000, Blood.

[10]  I. Correas,et al.  A constitutive region is responsible for nuclear targeting of 4.1R: modulation by alternative sequences results in differential intracellular localization. , 2000, Journal of cell science.

[11]  P. Taimen,et al.  Preferential expression of NuMA in the nuclei of proliferating cells. , 2000, Experimental cell research.

[12]  J. A. Gimm,et al.  Molecular and Functional Characterization of Protein 4.1B, a Novel Member of the Protein 4.1 Family with High Level, Focal Expression in Brain* , 2000, The Journal of Biological Chemistry.

[13]  R. Huganir,et al.  A Novel Neuron-Enriched Homolog of the Erythrocyte Membrane Cytoskeletal Protein 4.1 , 1999, The Journal of Neuroscience.

[14]  M. Snyder,et al.  A Nonerythroid Isoform of Protein 4.1R Interacts with the Nuclear Mitotic Apparatus (NuMA) Protein , 1999, The Journal of cell biology.

[15]  J. Conboy,et al.  Characterization of multiple isoforms of protein 4.1R expressed during erythroid terminal differentiation. , 1998, Blood.

[16]  S. Vetter,et al.  Characterization of a calcium-dependent calmodulin-binding domain in the 135-kD human protein 4.1 isoform. , 1998, European journal of biochemistry.

[17]  C. Tang,et al.  The 30-kD domain of protein 4.1 mediates its binding to the carboxyl terminus of pICln, a protein involved in cellular volume regulation. , 1998, Blood.

[18]  M. A. Alonso,et al.  An Alternative Domain Determines Nuclear Localization in Multifunctional Protein 4.1* , 1998, The Journal of Biological Chemistry.

[19]  S. Snyder,et al.  Cloning and characterization of 4.1G (EPB41L2), a new member of the skeletal protein 4.1 (EPB41) gene family. , 1998, Genomics.

[20]  S. Snyder,et al.  The 13-kD FK506 Binding Protein, FKBP13, Interacts with a Novel Homologue of the Erythrocyte Membrane Cytoskeletal Protein 4.1 , 1998, The Journal of cell biology.

[21]  N. Mohandas,et al.  Regulation of CD44-Protein 4.1 Interaction by Ca2+and Calmodulin , 1997, The Journal of Biological Chemistry.

[22]  C. Larabell,et al.  Structural Protein 4.1 in the Nucleus of Human Cells: Dynamic Rearrangements during Cell Division , 1997, The Journal of cell biology.

[23]  S. Karki,et al.  Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles , 1996, The Journal of cell biology.

[24]  K. Ramyar,et al.  A Complex of NuMA and Cytoplasmic Dynein Is Essential for Mitotic Spindle Assembly , 1996, Cell.

[25]  J. Conboy,et al.  Differential use of protein 4.1 translation initiation sites during erythropoiesis: implications for a mutation-induced stage-specific deficiency of protein 4.1 during erythroid development. , 1996, Blood.

[26]  D. Compton,et al.  NuMA assembles into an extensive filamentous structure when expressed in the cell cytoplasm. , 1996, Journal of cell science.

[27]  I. Correas,et al.  Protein 4.1 is a component of the nuclear matrix of mammalian cells. , 1995, The Biochemical journal.

[28]  C. Waterman-Storer,et al.  The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1). , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[29]  D. Branton,et al.  Identification of the Protein 4.1 Binding Interface on Glycophorin C and p55, a Homologue of the Drosophila discs-large Tumor Suppressor Protein (*) , 1995, The Journal of Biological Chemistry.

[30]  D. Branton,et al.  Cloning and characterization of hdlg: the human homologue of the Drosophila discs large tumor suppressor binds to protein 4.1. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[31]  E. Benz,et al.  Tissue-specific alternative splicing of protein 4.1 inserts an exon necessary for formation of the ternary complex with erythrocyte spectrin and F-actin. , 1993, Blood.

[32]  J. Conboy,et al.  Mechanochemistry of the alternatively spliced spectrin-actin binding domain in membrane skeletal protein 4.1. , 1993, The Journal of biological chemistry.

[33]  D. Compton,et al.  NuMA is required for the proper completion of mitosis , 1993, The Journal of cell biology.

[34]  T. Jöns,et al.  Identification of the binding interface involved in linkage of cytoskeletal protein 4.1 to the erythrocyte anion exchanger. , 1992, The EMBO journal.

[35]  I. Correas.  Characterization of isoforms of protein 4.1 present in the nucleus. , 1991, The Biochemical journal.

[36]  田中 敏彦.  Ca2＋－dependent Regulation of the Spectrin／Actin Interaction by Calmodulin and Protein 4.1 , 1991 .

[37]  P. Bertics,et al.  Phosphorylation of protein 4.1 on tyrosine-418 modulates its function in vitro. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[38]  J. Conboy,et al.  Tissue- and development-specific alternative RNA splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1. , 1991, The Journal of biological chemistry.

[39]  T. Tanaka,et al.  Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1. , 1991, The Journal of biological chemistry.

[40]  V. Marchesi,et al.  Heterogeneity of mRNA and protein products arising from the protein 4.1 gene in erythroid and nonerythroid tissues , 1990, The Journal of cell biology.

[41]  I. Correas,et al.  Erythrocyte protein 4.1 associates with tubulin. , 1988, The Biochemical journal.

[42]  V. Marchesi,et al.  Tissue‐specific analogues of erythrocyte protein 4.1 retain functional domains , 1988, Journal of cellular biochemistry.

[43]  D. Pettijohn,et al.  Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly. Properties of purified NuMA protein. , 1986, Experimental cell research.

[44]  T. Leto,et al.  Mechanisms of cytoskeletal regulation: Modulation of aortic endothelial cell protein band 4.1 by the extracellular matrix , 1986, Journal of cellular physiology.

[45]  J. Madri,et al.  Endothelial Cell‐Extracellular Matrix Interactions a , 1985, Annals of the New York Academy of Sciences.

[46]  B. L. Granger,et al.  Membrane skeletal protein 4.1 of avian erythrocytes is composed of multiple variants that exhibit tissue-specific expression , 1984, Cell.

[47]  C. M. Cohen,et al.  A protein immunologically related to erythrocyte band 4.1 is found on stress fibres of non-erythroid cells , 1982, Nature.

[48]  Samara L. Reck-Peterson,et al.  Nuclear actin and actin-related proteins in chromatin remodeling. , 2002, Annual review of biochemistry.

[49]  J. Conboy,et al.  Differentiation-associated switches in protein 4.1 expression. Synthesis of multiple structural isoforms during normal human erythropoiesis. , 1993, The Journal of clinical investigation.

[50]  E. Benz,et al.  Differential utilization of translation initiation sites in alternatively spliced mRNAs arising from the protein 4.1 gene. , 1992, Transactions of the Association of American Physicians.