Importance of class II transactivator leucine-rich repeats for dominant-negative function and nucleo-cytoplasmic transport.
Class II transactivator (CIITA), the master regulator of MHC class II (MHC-II) gene transcription, shows a complex behavior in terms of self-association, nucleo-cytoplasmic transport and MHC-II gene transactivation. Here, we analyzed the mechanisms of dominant-negative function and nucleo-cytoplasmic transport of CIITA with emphasis on the role of the C-terminal leucine-rich-repeat (LRR) region in these processes. First, we determined nucleo-cytoplasmic transport of endogenous CIITA and thus validated results obtained with epitope-tagged CIITA constructs. LRR mutations in potential protein-protein contact positions lead to either completely blocked or reduced nuclear import, but can also give rise to increased nuclear export. Surprisingly, N-terminally truncated CIITA mutants show dominant-negative inhibition of wild-type CIITA, whether they are located in the nucleus or in the cytoplasm. Integrity of the LRR is necessary for the dominant-negative function of both types of mutants. LRR mutations are dominant over the effect of an exogenously added N-terminal nuclear localization signal (NLS) leading to cytoplasmic localization. Taken together, our results show that the LRR regulate the function of one or several NLS within CIITA, and control both nuclear import and export. Self-association is not affected in these mutants; we therefore suggest that interaction of the LRR with an unknown protein partner may be necessary for import and transactivation function of CIITA.
Camacho-Carvajal
Margarita M
MM
Hans-Spemann-Laboratories, Max-Planck-Institut für Immunbiologie, Freiburg D-79108, Germany.
Klingler
Sebastian
S
Schnappauf
Felix
F
Hake
Sandra B
SB
Steimle
Viktor
V
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D021381:Protein Transport / Q000502:physiology*; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology; D014162:Transfection
C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D015534:Trans-Activators; D007930:Leucine