Functional characterization of the microtubule-binding and -destabilizing domains of CPAP and d-SAS-4.
We previously identified a novel centrosomal protein CPAP, which carries a 112-residue motif that is essential for microtubule destabilization. In this report, we define both the microtubule (MT) binding and destabilizing domains in human CPAP and analyze the mutations that affect its MT-destabilizing activity. Analysis of a series of CPAP truncated proteins showed that the MT-binding domain (MBD; residues 423-607) of CPAP is located next to its MT-destabilizing domain (MDD; residues 311-422). Site-specific mutagenesis revealed that the mutations that either disrupt the alpha-helical structure (Y341P, I346P, L348P, and triple-P) or alter the charge property (KR377EE) of the MDD significantly affect its MT-destabilizing ability. The activity for binding to a tubulin heterodimer was also significantly reduced in KR377EE mutant. Furthermore, we have analyzed the putative function of Drosophila d-SAS-4, a distant relative of human CPAP, which shares a conserved approximately 20-aa sequence with the MDD of CPAP. Our results show that mutations in this conserved sequence also eliminate d-SAS-4's MT-destabilizing activity, suggesting that d-SAS-4 and CPAP may play similar roles within cells.
Hsu
Wen-Bin
WB
Institute of Biomedical Sciences, Academia Sinica, 128 Academia Road, Sec. 2, Taipei 11529, Taiwan.
Hung
Liang-Yi
LY
Tang
Chieh-Ju C
CJ
Su
Chia-Li
CL
Chang
Yulin
Y
Tang
Tang K
TK
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D019281:Dimerization; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014404:Tubulin / Q000378:metabolism
C414735:CENPJ protein, human; D029721:Drosophila Proteins; D008869:Microtubule-Associated Proteins; D050505:Mutant Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C522063:Sas-4 protein, Drosophila; D014404:Tubulin; D049452:Green Fluorescent Proteins; D017239:Paclitaxel