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.
Mattagajasingh
Subhendra N
SN
Cardiovascular Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
Huang
Shu-Ching
SC
Benz
Edward J
EJ
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D034521:Nuclear Matrix-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D034521:Nuclear Matrix-Associated Proteins; D011993:Recombinant Fusion Proteins; C028841:erythrocyte membrane band 4.1 protein; D049452:Green Fluorescent Proteins; D000409:Alanine