NFATc1/C harbors three SUMO consensus motifs which facilitate
interaction with Ubc9. A, schematic representation of NFATc1
isoforms and the SUMO consensus motifs (Ψ-Lys-X-Glu; Ψ=
isoleucine/leucine/valine) shown by arrows. The short isoforms harbor
only the common Lys-349 site, whereas NFATc1/C contains sites at Lys-702 and
-914 in addition. Lysines to arginine exchanges create ΔSUMO mutations.
The mutation of both C-terminal lysines is designated as K702R/K914R and of
all three sites as K349R/K702R/K914R. An indication for the B-term and CC-term
peptides are given. TAD, transactivation domain; RSD, Rel
similarity domain; NLS, nuclear localization signal; NES,
nuclear export signal; SRR and SP, Ser/Thr phosphorylation sites,
respectively. B, NFATc1/C physically interacts with Ubc9 in
vivo. HA-tagged c1/C-coding vector was transfected into 293T HEK cells,
either in combination with pcDNA expressing FLAG-tagged Ubc9 or FLAG only.
After 6 h of stimulation with T/I and a total of 48 h, protein lysates were
prepared and subjected to anti-FLAG IP followed by immunoblot detection with
anti-HA. Whole cell lysates (WCL) were also directly analyzed by
immunoblotting with anti-HA and anti-FLAG, respectively. C, the SUMO
site-deficient mutant K349R/K702R/K914R cannot recruit Ubc9. Procedures were
the same as in B, except that K349R/K702R/K914R was included.
D, the SUMO site at Lys-702 is the most relevant. Procedures were the
same as in C but including all different SUMO site deficient mutants.
E, the C terminus of NFATc1/C is sufficient for binding Ubc9.
Procedures were the same as before, but EYFP-tagged NFATc1/C or only the C
terminus were analyzed. F, both the NFATc1/B specific
(B-term) and the NFATc1/C only (CC-term) C termini interact
with Ubc9. Procedures were as before, but by use of constructs with nuclear
localization signal-estrogen receptor fusions (estrogen receptor α),
which are released for nuclear localization by addition of 4-hydroxytamoxifen.
*, heavy or light chain of the precipitation antibody.