Correction
for Ohyama et al., J. Neurosci. 22 (9) 3342-3351.
Previous Article
The Journal of Neuroscience, July 1, 2002, 22(13):5789-5789
CORRECTION
In the article, "Regulation of Exocytosis through
Ca2+/ATP-Dependent Binding of Autophosphorylated
Ca2+/Calmodulin-Activated Protein Kinase II to Syntaxin
1A," by Akihiro Ohyama, Kohei Hosaka, Yoshiaki Komiya, Kimio Akagawa,
Emiko Yamauchi, Hisaaki Taniguchi, Nobuyuki Sasagawa,
Ko- nosuke Kumakura, Sumiko Mochida, Takashi Yamauchi,
and Michihiro Igarashi, which appeared on pages 3342-3351 of the May
1, 2002 issue, Figure 2B,E printed with several labels
missing. A revised version of Figure 2, along with a corrected legend,
is printed here.
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Figure 2.
CaMKII binds to syntaxin only when
autophosphorylated. A, Purified CaMKII also binds
syntaxin after autophosphorylation. The fraction unbound to
GST syntaxin after re-autophosphorylation was not recognized by
anti-autophosphorylated CaMKII antibody. CaMKII (5 µg) purified
from rat brain was autophosphorylated (Auto-P) in buffer
containing 50 mM HEPES-NaOH, pH 8.0, 8 mM Mg
(CH3COO)2, 0.25 mM
CaCl2, 2 µM CaM, and 0.5 mM ATP
for 5 min at 30°C, and then incubated with immobilized GST or
GSTsyntaxin (4 nmol) for 1 hr at 4°C. After centrifugation, the
supernatant was collected as the unbound fraction. The PreScission
protease fraction was collected as the bound fraction. Samples were
resolved by 10% SDS-PAGE and immunoblotted using anti-CaMKII mAb or
anti-autophosphorylated CaMKII mAb. B, T286A-CaMKII could
not bind syntaxin. Top panel (two lanes), Both
wild-type CaMKII (wt) and T286A-CaMKII (T286A)
were produced by in vitro translation. Their apparent
molecular masses wre ~48 kDa. Bottom panel (four
lanes), Wild-type CaMKII (Syx+wt), but not
T286-CaMKII (Syx+T286A), bound to syntaxin 1A in a
pull-down study in the presence of Ca2+/ATP. The wild-type
CaMKII did not bind to GST (GST+wt). Just as seen for the
native CaMKII from rat brain (Fig. 1B), the wild-type
CaMKII produced by in vitro translation could not bind to
syntaxin without Ca2+ (Syx+WT Ca). The cDNA
encoding rat CaMKII or T286A-CaMKII (provided by Dr. H. Schulman)
was added to the in vitro translation kit (Promega).
Proteins were expressed by incubating kit components for 1.5 hr at
30°C and then by adding immobilized GSTsyntaxin as described above.
After elution with SDS-sample buffer, bound proteins were blotted and
detected using streptavidin-conjugated alkaline phosphatase. Molecular
masses (in kilodaltons) are shown to the left. C, CaMKII
produced using in vitro translation also shows Ca2+
sensitivity for binding to syntaxin after autophosphorylation.
Translation in vitro proceeded as described above, and
CaMKII was autophosphorylated in buffer containing Tris-HCl, pH 7.6, 0.5 mM CaCl2, 2 µM CaM, 2 mM MgCl2, and 0.5 mM ATP at 30°C
for 15 min. The autophosphorylated CaMKII was incubated with
immobilized GSTsyntaxin (4 nmol) at 4°C for 1 hr and then with
binding buffer containing various concentrations of Ca2+
for an additional 1 hr. D, Dose-dependent binding of CaMKII
to syntaxin. GSTsyntaxin (4 nmol) was incubated with recombinant
CaMKII at various concentrations. E, CaMKII lacking
the association domain [1-325] (i.e., monomeric CaMKII) does not
bind to syntaxin, even when autophosphorylated. Non-autophosphorylated
[Auto-P (), Gst-syn 1A (+)] or autophosphorylated
monomeric CaMKII [(Auto-P (+), GST-syn 1A(+)] was
incubated with GSTsyntaxin 1A, and each bound fraction was eluted as
described above (see A). Together with these fractions, both
forms of monomeric CaMKII before incubation with the immobilized
syntaxin [Auto-P (), GST-syn 1A () or Auto-P (+),
GST-syn 1A ()] were also electrophoreses and analyzed by
immonoblotting. The apparent molecular mass of CaMKII [1-325] was
approximately 35 kDa and was recognized by anti-CaMKII mAb. After
the autophosphorylation, the truncated CaMKII was also recognized by
anti-P-Thr286 CaMKII mAb, as well as the native one (see
A).
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Copyright © 2002 Society for Neuroscience 0270-6474/02/22135789-01$05.00/0
