Calmodulin-dependent nuclear import of HMG-box family nuclear factors: importance of the role of SRY in sex reversal.
The HMG (high-mobility group)-box-containing chromatin-remodelling factor SRY (sex-determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus is critically dependent on two NLSs (nuclear localization signals) that flank its HMG domain: the C-terminally located 'beta-NLS' that mediates nuclear transport through Impbeta1 (importin beta1) and the N-terminally located 'CaM-NLS' which is known to recognize the calcium-binding protein CaM (calmodulin). In the present study, we examined a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localization of GFP (green fluorescent protein)-SRY fusion proteins in transfected cells compared with wild-type. The CaM antagonist CDZ (calmidazolium chloride) was found to significantly reduce wild-type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ's effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM-binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG-box-domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found for the first time to rely on CaM for optimal nuclear localization. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors.
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Gurpreet
G
Kaur
Aurelie
A
Delluc-Clavieres
Ivan K H
IK
Poon
Jade K
JK
Forwood
Dominic J
DJ
Glover
David A
DA
Jans
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D012734:Disorders of Sex Development; D005260:Female; D006061:Gonadal Dysgenesis, 46,XY / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D024241:HMGN1 Protein / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D020125:Mutation, Missense; D019913:Nuclear Localization Signals; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051876:Sex-Determining Region Y Protein / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism
D002147:Calmodulin; D024241:HMGN1 Protein; D007093:Imidazoles; C424166:KPNB1 protein, human; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D051876:Sex-Determining Region Y Protein; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins; C031938:calmidazolium