Requirement of PKR dimerization mediated by specific hydrophobic residues for its activation by double-stranded RNA and its antigrowth effects in yeast

被引:47
作者
Patel, RC [1 ]
Sen, GC [1 ]
机构
[1] Cleveland Clin Fdn, Dept Mol Biol, Lerner Res Inst, Cleveland, OH 44195 USA
关键词
D O I
10.1128/MCB.18.12.7009
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The roles of protein dimerization and double-stranded RNA (dsRNA) binding in the biochemical and cellular activities of PKR, the dsRNA-dependent protein kinase, were investigated. We have previously shown that both properties of the protein are mediated by the same domain. Here we show that dimerization is mediated by hydrophobic residues present on one side of an amphipathic oc-helical structure within this domain. Appropriate substitution mutations of residues on that side produced mutants with increased or decreased dimerization activities. Using these mutants, we demonstrated that dimerization is not essential for dsRNA binding. However, enhancing dimerization artificially, by providing an extraneous dimerization domain, increased dsRNA binding of both wild-type and mutant proteins. In vitro, the dimerization-defective mutants could not be activated by dsRNA but were activated normally by heparin. In Saccharomyces cerevisiae, unlike wild-type PKR, these mutants could not inhibit cell growth and the dsRNA-binding domain of the dimerization-defective mutants could not prevent the antigrowth effect of wild-type PKR. These results demonstrate the biological importance of the dimerization properties of PKR.
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页码:7009 / 7019
页数:11
相关论文
共 66 条
[1]   DETECTION OF PROTEIN-KINASE HOMOLOGS AND VIRAL RNA-BINDING DOMAINS UTILIZING POLYCLONAL ANTISERUM PREPARED AGAINST A BACULOVIRUS-EXPRESSED DS RNA-ACTIVATED 68,000-DA PROTEIN-KINASE [J].
BARBER, GN ;
TOMITA, J ;
GARFINKEL, MS ;
MEURS, E ;
HOVANESSIAN, A ;
KATZE, MG .
VIROLOGY, 1992, 191 (02) :670-679
[2]   THE 58-KILODALTON INHIBITOR OF THE INTERFERON-INDUCED DOUBLE-STRANDED RNA-ACTIVATED PROTEIN-KINASE IS A TETRATRICOPEPTIDE REPEAT PROTEIN WITH ONCOGENIC PROPERTIES [J].
BARBER, GN ;
THOMPSON, S ;
LEE, TG ;
STROM, T ;
JAGUS, R ;
DARVEAU, A ;
KATZE, MG .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (10) :4278-4282
[3]  
BISCHOFF JR, 1985, J BIOL CHEM, V260, P8237
[4]   THE CELLULAR 68,000-MR PROTEIN-KINASE IS HIGHLY AUTOPHOSPHORYLATED AND ACTIVATED YET SIGNIFICANTLY DEGRADED DURING POLIOVIRUS INFECTION - IMPLICATIONS FOR TRANSLATIONAL REGULATION [J].
BLACK, TL ;
SAFER, B ;
HOVANESSIAN, A ;
KATZE, MG .
JOURNAL OF VIROLOGY, 1989, 63 (05) :2244-2251
[5]   NMR SOLUTION STRUCTURE OF A DSRNA BINDING DOMAIN FROM DROSOPHILA STAUFEN PROTEIN REVEALS HOMOLOGY TO THE N-TERMINAL DOMAIN OF RIBOSOMAL-PROTEIN S5 [J].
BYCROFT, M ;
GRUNERT, S ;
MURZIN, AG ;
PROCTOR, M ;
STJOHNSTON, D .
EMBO JOURNAL, 1995, 14 (14) :3563-3571
[6]   AN AMINO-TERMINAL FRAGMENT OF GAL4 BINDS DNA AS A DIMER [J].
CAREY, M ;
KAKIDANI, H ;
LEATHERWOOD, J ;
MOSTASHARI, F ;
PTASHNE, M .
JOURNAL OF MOLECULAR BIOLOGY, 1989, 209 (03) :423-432
[7]  
Carpick BW, 1997, J BIOL CHEM, V272, P9510, DOI 10.1074/jbc.272.14.9510
[8]   HUMAN P68 KINASE EXHIBITS GROWTH SUPPRESSION IN YEAST AND HOMOLOGY TO THE TRANSLATIONAL REGULATOR GCN2 [J].
CHONG, KL ;
FENG, L ;
SCHAPPERT, K ;
MEURS, E ;
DONAHUE, TF ;
FRIESEN, JD ;
HOVANESSIAN, AG ;
WILLIAMS, BRG .
EMBO JOURNAL, 1992, 11 (04) :1553-1562
[9]   STRUCTURE AND REGULATION OF EUKARYOTIC INITIATION-FACTOR EIF-2 - SEQUENCE OF THE SITE IN THE ALPHA-SUBUNIT PHOSPHORYLATED BY THE HEME-CONTROLLED REPRESSOR AND BY THE DOUBLE-STRANDED RNA-ACTIVATED INHIBITOR [J].
COLTHURST, DR ;
CAMPBELL, DG ;
PROUD, CG .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1987, 166 (02) :357-363
[10]   DOUBLE-STRANDED-RNA-DEPENDENT PROTEIN-KINASE AND TAR RNA-BINDING PROTEIN FORM HOMODIMERS AND HETERODIMERS IN-VIVO [J].
COSENTINO, GP ;
VENKATESAN, S ;
SERLUCA, FC ;
GREEN, SR ;
MATHEWS, MB ;
SONENBERG, N .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (21) :9445-9449