Leptomycin B (LMB), a specific inhibitor of CRM1-mediated nuclear export18
induced a rapid nuclear accumulation of endogenous DARPP-32 () or transfected D32-GFP (Supp. Fig.5a
) in virtually all neurons. We identified at residues 103-111 of DARPP-32 a putative nuclear export signal (NES, ), homologous to sequences known to bind CRM119
. Mutation of Leu-103 or Leu-109 to alanine induced a dramatic accumulation of DARPP-32-GFP in the nucleus (), confirming the critical role of this sequence in nuclear export. Similar results were observed in CHO cells (Supp. Fig.5b,c
). We also found that the N-terminal region of DARPP-32, which encompasses several putative nuclear localization signals, played a critical role in its nuclear import (data not shown).
DARPP-32 undergoes a continuous cyto-nuclear shuttling
These results suggested that D1R stimulation acted by reducing the export of DARPP-32 out of the nucleus. We ruled out a general inhibitory influence of D1R stimulation on CRM1-dependent nuclear export, by showing that SKF81297 did not alter the localization of another protein with an active NES20
/calmodulin-dependent kinase-Iα (CaMKIα-GFP, ). Introduction of an ectopic NES sequence derived from prohibitin21
) at the C-terminus of DARPP-32 (D32-NESproh
-GFP) blocked the translocation induced by SKF81297 ().
To determine how the D1R/cAMP pathway regulated DARPP-32 localization we mutated its major phosphorylation sites (Thr-34, Thr-75, Ser-97, and Ser-130, ) to alanine. In striatal neurons in culture, basal and D1R-induced localization of T34A-, T75A- and S130A-DARPP-32-GFP mutants was similar to that of wild type protein (Supp. Fig.6a
). In support of these observations, in knock-in mutant mice bearing an alanine point mutation at Thr-34 or Thr-75 the basal localization of DARPP-32 and its cocaine-induced nuclear accumulation was similar to wild type (Supp. Fig.6b,c
). In contrast, when Ser-97, located close to the NES of DARPP-32, was mutated to alanine, the protein was nuclear in basal conditions and D1R stimulation did not alter this distribution (S97A-D32-GFP, ). Mutation of Ser-97 to phospho-mimic acidic residues, glutamate (S97E) or aspartate (S97D) induced a preferential cytoplasmic localization, which was not modified by SKF81297 (, Supp. Fig.7a
Phosphorylation of Ser-97 controls intracellular localization of DARPP-32
Ser-97 (Ser-102 in rat) is highly phosphorylated in basal conditions by casein kinase-2 (CK2)22
. An inhibitor of CK2, 4,5,6,7-tetrabromobenzotriazole (TBB)23
, increased markedly nuclear DARPP-32-GFP, and prevented the effects of D1R-agonist treatment (, Supp. Fig.7b
). The effects of TBB resulted from prevention of Ser-97 phosphorylation, since they were not observed with S97E-D32-GFP (, Supp. Fig.7c
). These results supported the hypothesis that DARPP-32 is mainly nuclear when Ser-97 is dephosphorylated, whereas it is preferentially cytoplasmic when phosphorylated by CK2.