Leucine-rich repeats of the class II transactivator control its rate of nuclear accumulation.
Activation of class II major histocompatibility complex (MHC) gene expression is regulated by a master regulator, class II transcriptional activator (CIITA). Transactivation by CIITA requires its nuclear import. This study will address a mechanistic role for the leucine-rich repeats (LRR) of CIITA in regulating nuclear translocation by mutating 12 individual consensus-motif "leucine" residues in both its alpha-motifs and beta-motifs. While some leucine mutations in the LRR motif of CIITA cause congruent loss of transactivation function and nuclear import, other alanine substitutions in both the alpha-helices and the beta-sheets have normal transactivation function but a loss of nuclear accumulation (i.e., functional mutants). This seeming paradox is resolved by the observations that nuclear accumulation of these functional mutants does occur but is significantly less than wild-type. This difference is revealed only in the presence of leptomycin B and actinomycin D, which permit examination of nuclear accumulation unencumbered by nuclear export and new CIITA synthesis. Further analysis of these mutants reveals that at limiting concentrations of CIITA, a dramatic difference in transactivation function between mutants and wild-type CIITA is easily detected, in agreement with their lowered nuclear accumulation. These experiments reveal an interesting aspect of LRR in controlling the amount of nuclear accumulation.
Harton
Jonathan A
JA
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.
O'Connor
William
W
Conti
Brian J
BJ
Linhoff
Michael W
MW
Ting
Jenny P-Y
JP
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000949:Histocompatibility Antigens Class II; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D007930:Leucine