Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1
ITSN1 is an endocytic scaffold protein with a prominent function in synaptic transmission. It is known that Ca signaling is crucial for the regulation of synaptic proteins functioning. Aim. Checking the possibility of Ca/calmodulin-dependent phosphorylation of ITSN1. Methods. Affinity chromatography...
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Інститут молекулярної біології і генетики НАН України
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| Zitieren: | Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 / D.Ye. Morderer, O.V. Nikolaienko, I.Ya. Skrypkina, O.V. Rymarenko, S.V. Kropyvko, L.O. Tsyba, A.V. Rynditch // Вiopolymers and Cell. — 2014. — Т. 30, № 1. — С. 74-76. — Бібліогр.: 11 назв. — англ. |
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irk-123456789-1537312019-06-15T01:32:05Z Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 Morderer, D.Ye. Nikolaienko, O.V. Skrypkina, I.Ya. Rymarenko, O.V. Kropyvko, S.V. Tsyba, L.O. Rynditch, A.V. Short Communications ITSN1 is an endocytic scaffold protein with a prominent function in synaptic transmission. It is known that Ca signaling is crucial for the regulation of synaptic proteins functioning. Aim. Checking the possibility of Ca/calmodulin-dependent phosphorylation of ITSN1. Methods. Affinity chromatography, in vitro kinase reaction, Western blotting, gel staining with fluorescent stains. Results. We show that the fraction of calmodulin-binding proteins is able to phosphorylate the recombinant fragments encoding the coiled-coil region and the SH3 domain-containing region of ITSN1 in the presence of Ca ions and calmodulin. Conclusions. The coiled-coil region and the SH3 domain-containing region of ITSN1 undergo Ca/calmodulin-dependent phosphorylation in vitro, suggesting a possible regulation of ITSN1 by Ca signaling. ITSN1 – це ендоцитозний адапторний білок, що виконує значущі функції у синаптичному передаванні нервового імпульсу. Відомо, що кальцієва сигналізація є ключовим елементом регуляції функціонування синаптичних білків. Мета. Дослідити можливість кальцій/кальмодулін-залежного фосфорилювання ITSN1. Методи. Афінна хроматографія, кіназна реакція in vitro, Вестерн блот-гібридизація, фарбування гелів флуоресцентними барвниками. Результати. Показано, що фракція кальмодулін-зв’язувальних білків здатна фосфорилювати рекомбінантні надспіралізовану та SH3 домен-вмісну ділянки ITSN1 in vitro за присутності іонів кальцію і кальмодуліну. Висновки. Надспіралізована та SH3 домен-вмісна ділянки ITSN1 підлягають кальцій/кальмодулін-залежному фосфорилюванню in vitro, що дозволяє припустити існування регуляції ITSN1 кальцієвою сигнальною системою. ITSN1 –это эндоцитозный адапторный белок, выполняющий значительные функции в синаптической передаче нервного импульса. Известно, что кальциевая сигнализация является ключевым элементом регуляции функционирования синаптических белков. Цель. Исследовать возможность кальций/кальмодулин-зависимого фосфорилирования ITSN1. Методы. Аффинная хроматография, киназная реакция in vitro, Вестерн блот-гибридизация, окрашивание белков флуоресцентными красителями. Результаты. Показано, что фракция кальмодулин-связывающих белков способна фосфорилировать рекомбинантные суперспирализованный и SH3 домен-содержащий участки ITSN1 in vitro в присутствии ионов кальция и кальмодулина. Выводы. Cуперспирализованный и SH3 домен-содержащий участки ITSN1 подлежат кальций/кальмодулин-зависимому фосфорилированию in vitro, что позволяет предположить существование регуляции ITSN1 кальциевой сигнальной системой. 2014 Article Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 / D.Ye. Morderer, O.V. Nikolaienko, I.Ya. Skrypkina, O.V. Rymarenko, S.V. Kropyvko, L.O. Tsyba, A.V. Rynditch // Вiopolymers and Cell. — 2014. — Т. 30, № 1. — С. 74-76. — Бібліогр.: 11 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000882 http://dspace.nbuv.gov.ua/handle/123456789/153731 577.336+667.287.4 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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English |
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Short Communications Short Communications |
| spellingShingle |
Short Communications Short Communications Morderer, D.Ye. Nikolaienko, O.V. Skrypkina, I.Ya. Rymarenko, O.V. Kropyvko, S.V. Tsyba, L.O. Rynditch, A.V. Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 Вiopolymers and Cell |
| description |
ITSN1 is an endocytic scaffold protein with a prominent function in synaptic transmission. It is known that Ca signaling is crucial for the regulation of synaptic proteins functioning. Aim. Checking the possibility of Ca/calmodulin-dependent phosphorylation of ITSN1. Methods. Affinity chromatography, in vitro kinase reaction, Western blotting, gel staining with fluorescent stains. Results. We show that the fraction of calmodulin-binding proteins is able to phosphorylate the recombinant fragments encoding the coiled-coil region and the SH3 domain-containing region of ITSN1 in the presence of Ca ions and calmodulin. Conclusions. The coiled-coil region and the SH3 domain-containing region of ITSN1 undergo Ca/calmodulin-dependent phosphorylation in vitro, suggesting a possible regulation of ITSN1 by Ca signaling. |
| format |
Article |
| author |
Morderer, D.Ye. Nikolaienko, O.V. Skrypkina, I.Ya. Rymarenko, O.V. Kropyvko, S.V. Tsyba, L.O. Rynditch, A.V. |
| author_facet |
Morderer, D.Ye. Nikolaienko, O.V. Skrypkina, I.Ya. Rymarenko, O.V. Kropyvko, S.V. Tsyba, L.O. Rynditch, A.V. |
| author_sort |
Morderer, D.Ye. |
| title |
Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 |
| title_short |
Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 |
| title_full |
Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 |
| title_fullStr |
Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 |
| title_full_unstemmed |
Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 |
| title_sort |
ca/calmodulin-dependent phosphorylation of endocytic scaffold itsn1 |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2014 |
| topic_facet |
Short Communications |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/153731 |
| citation_txt |
Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1 / D.Ye. Morderer, O.V. Nikolaienko, I.Ya. Skrypkina, O.V. Rymarenko, S.V. Kropyvko, L.O. Tsyba, A.V. Rynditch // Вiopolymers and Cell. — 2014. — Т. 30, № 1. — С. 74-76. — Бібліогр.: 11 назв. — англ. |
| series |
Вiopolymers and Cell |
| work_keys_str_mv |
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| first_indexed |
2025-07-14T05:13:03Z |
| last_indexed |
2025-07-14T05:13:03Z |
| _version_ |
1837597966353825792 |
| fulltext |
74
UDC 577.336+667.287.4
Ca2+/calmodulin-dependent phosphorylation
of endocytic scaffold ITSN1
D. Ye. Morderer, O. V. Nikolaienko, I. Ya. Skrypkina, O. V. Rymarenko,
S. V. Kropyvko, L. O. Tsyba, A. V. Rynditch
State Key Laboratory of Molecular and Cellular Biology,
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
dmytromorderer@gmail.com
ITSN1 is an endocytic scaffold protein with a prominent function in synaptic transmission. It is known that Ca2+
signaling is crucial for the regulation of synaptic proteins functioning. Aim. Checking the possibility of
Ca2+/calmodulin-dependent phosphorylation of ITSN1. Methods. Affinity chromatography, in vitro kinase
reaction, Western blotting, gel staining with fluorescent stains. Results. We show that the fraction of calmodu-
lin-binding proteins is able to phosphorylate the recombinant fragments encoding the coiled-coil region and the
SH3 domain-containing region of ITSN1 in the presence of Ca2+ ions and calmodulin. Conclusions. The coiled-
coil region and the SH3 domain-containing region of ITSN1 undergo Ca2+/calmodulin-dependent phospho-
rylation in vitro, suggesting a possible regulation of ITSN1 by Ca signaling.
Keywords: ITSN1, phosphorylation, calmodulin, Ca2+ signaling.
Introduction. ITSN1 is a scaffold protein involved in
endocytosis and signal transduction. This protein pos-
sesses several domains that act as platforms for binding
molecular partners (Fig. 1). To date ITSN1 has been
shown to participate in numerous interactions with
plenty of proteins [1–3]. The number of its known bin-
ding partners is much higher than the quantity of its
modules for binding. Thus, it is reasonable to suggest
that some regulatory mechanisms should exist in order
to determine the sets of ITSN1-interacting partners in
particular physiological contexts.
One of such mechanisms can be provided by the
posttranslational modifications (PTM) which may alter
binding properties of ITSN1 and thus adjust its func-
tion to current requirements. Phosphorylation is the most
common and most studied type of PTM to date. The
phosphorylation of ITSN1 was shown previously by
large-scale mass spectrometry studies [4]. Moreover,
recently the tyrosine phosphorylation of ITSN1 pro-
moted by expression of LMP2A protein of Epstein-Barr
virus has been reported [5]. However, neither the phy-
siological background nor functional consequences of
these phosphorylation events are known.
ITSN1 is an important player in synaptic vesicle cyc-
ling [6]. It is widely accepted that the majority of mole-
cular events associated with the synaptic vesicle traffic-
king are triggered by changes in Ca2+ concentration and
subsequent activation of the Ca2+-dependent proteins,
particularly kinases and phosphatases [7]. In this work
we tested the hypothesis that ITSN1 may undergo the
Ca2+/calmodulin-dependent phosphorylation. We report
that in in vitro conditions at least two structural compo-
nents of ITSN1, namely the coiled-coil region and the
SH3 domain-containing region, can be phosphorylated
in the Ca2+/calmodulin-dependent manner.
Materials and methods. Expression constructs and
antibodies. GST-SH3A-E plasmid was described pre-
viously [5]. GST-CCR was obtained by cloning cDNA
fragment encoding the coiled-coil region of ITSN1 (aa
323–726, accession number Q15811 in UniProtKB) in
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 1. P. 74–76 doi: http://dx.doi.org/10.7124/bc.000882
Ó Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
pGEX-4T-3 vector («GE Healthcare», UK). The mouse
monoclonal anti-STOP antibodies (MAB5524) and the se-
condary horseradish peroxidase-labeled (HRP) goat anti-
mouse antibodies were purchased from «Millipore»
(USA) and «Promega» (USA), respectively.
Isolation of calmodulin-binding proteins. Mouse
brains were homogenized in MEM buffer (100 mM
MOPS, pH 6.75, 400 mM NaCl, 1 mM MgCl2, 1 mM
EGTA, 2 mM DTT and complete EDTA-free protease
inhibitor cocktail («Roche», France)) and centrifuged
for 20 min at 12,000 g at 4 °C. Supernatant was suppli-
ed by CaCl2 to the final concentration of 2 mM and loa-
ded to the column filled by calmodulin-agarose («Sig-
ma-Aldrich», USA). After washing with the same buf-
fer, bound proteins were eluted from the column by MEM
buffer, which did not contain CaCl2.
In vitro kinase reaction. Isolated calmodulin-bin-
ding proteins were dialyzed against kinase buffer (50
mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT)
using Spectra/Por 1 Dialysis Membrane («Spectrum La-
boratories Inc.», USA). Recombinant GST and GST-fu-
sed proteins were expressed in Escherichia coli BL21
(DE3) and purified on glutathione sepharose 4B beads
(«GE Healthcare») according to manufacturer’s in-
structions. After purification, proteins bound on sepha-
rose beads were washed in kinase buffer and supplied
by dialyzed calmodulin-binding protein fraction to the
final volume of 50 µl in each reaction. All the reactions
contained 0.4 mM EGTA and 0.5 µM microcystine.
Additionally, some of the samples contained 100 µM
ATP, 3 µM calmodulin, 2 mM CaCl2 and 2 mM EGTA.
Reactions were performed at 30 °C for 30 min and
stopped by adding equal volume of Laemmli sample
buffer (150 mM Tris-HCl, pH 6.8, 2.5 % glycerol, 10 %
SDS, 3 % b-mercaptoethanol and 0.5 % bromophenol
blue). Then the samples were boiled, resolved by
SDS-PAGE and either transferred to a nitrocellulose
membrane for Western-blot analysis, or subjected to
staining with Pro-Q Diamond or SYPRO Ruby protein
stains («Molecular Probes», USA) according to manu-
facturer’s instructions. Stained gels were analyzed on
PharosFX Molecular Imager («BioRad», USA), images
of Western-blots were acquired on ChemiDocTM XRS+
system («BioRad»).
Results and discussion. In order to examine the pos-
sibility of Ca2+/calmodulin-dependent ITSN1 phospho-
rylation we purified calmodulin-dependent kinases along
with other calmodulin-binding proteins from the mou-
se brain protein lysate by affinity chromatography using
calmodulin-agarose. The efficiency of purification was
checked by protein staining with SYPRO Ruby stain
and by Western blotting of calmodulin-binding protein
STOP (stable tubule-only polypeptide) (Fig. 2, A). The
obtained purified proteins were used as a source of the
kinase activity for in vitro kinase assay with the GST-
fused coiled-coil region (CCR) and the SH3-domain-
containing fragment (SH3A-E) of ITSN1. Addition of
Ca2+ ions and calmodulin to the reaction resulted in the
phosphorylation of both GST-fused CCR and SH3A-E
proteins visualized by Pro-Q Diamond Phosphoprotein
Gel Stain (Fig. 2, B). No phosphorylation was observed
without Ca and calmodulin. GST alone was equally stai-
ned in all the reactions, including a negative control,
which indicates the absence of phosphorylation. Uni-
formity of protein loading was controlled by post-stai-
ning the gels with SYPRO Ruby (data not shown).
Our results for the first time demonstrate the Ca2+/
calmodulin-dependent phosphorylation of ITSN1, rai-
sing the question of its regulation by Ca2+ signaling. It
has been shown previously that ITSN1 undergoes acti-
vity dependent shuttling between active and periactive
zones in presynaptic termini [8, 9], but the way of con-
version of electrical stimulation to such molecular
event remains unknown. Taking into account the
crucial role of Ca2+ in synaptic activity, the Ca2+/
calmodulin phosphorylation of ITSN1 may occur in
response to synaptic stimulation and result in alteration
of its interaction profile which may lead to the changes
in its functioning. Furthermore, ITSN1 was shown to
participate in dendritic spine development [10] that can
also be regulated by Ca2+ signaling since the Ca2+/
calmodulin-dependent kinases are known to be the
important players in neuronal de velopment and
synaptic plasticity [11]. The precise sites of
phosphorylation and its effect on the ITSN1 structure
75
CA/CALMODULIN-DEPENDENT PHOSPHORYLATION OF ENDOCYTIC SCAFFOLD ITSN1
ITSN1
EH
domains
CCR SH3 domains DH-PH-C2
domains
Fig. 1. Schematic representation of ITSN1 domain organization. Do-
mains used for the experiments are shown in bold
and functions are the focus of our further
investigations.
Conclusions. The coiled-coil region and the SH3
domain-containing region of ITSN1 undergo the Ca2+/
calmodulin-dependent phosphorylation in vitro.
Êàëüö³é/êàëü ìî äóë³í-çà ëåæ íå ôîñ ôî ðè ëþ âàí íÿ
åí äî öè òîç íî ãî àäàï òî ðà ITSN1
Ä. ª. Ìîð äå ðåð, Î. Â. ͳêîëàºíêî, ². ß. Ñêðèïê³íà,
Î. Â. Ðè ìà ðåí êî, Ñ. Â. Êðî ïèâ êî, Ë. Î. Öèáà, À. Â. Ðèí äè÷.
ITSN1 – öå åí äî öè òîç íèé àäàï òîð íèé á³ëîê, ùî âè êî íóº çíà ÷óù³
ôóíêö³¿ ó ñè íàï òè÷ íî ìó ïå ðå äà âàíí³ íå ðâî âî ãî ³ìïóëü ñó. ³äîìî,
ùî êàëüö³ºâà ñèã íàë³çàö³ÿ º êëþ ÷î âèì åëå ìåí òîì ðå ãó ëÿö³¿ ôóíê-
ö³îíó âàí íÿ ñè íàï òè÷ íèõ á³ëê³â. Ìåòà. Äîñë³äèòè ìîæ ëèâ³ñòü
êàëüö³é/êàëü ìî äóë³í-çà ëåæ íî ãî ôîñ ôî ðè ëþ âàí íÿ ITSN1. Ìå òî -
äè. Àô³ííà õðî ìà òîã ðàô³ÿ, ê³íà çíà ðå àêö³ÿ in vitro, Âåñ òåðí áëîò-
ã³áðèäè çàö³ÿ, ôàð áó âàí íÿ ãåë³â ôëó î ðåñ öåí òíè ìè áàð âíè êà ìè. Ðå-
çóëü òà òè. Ïîêàçà íî, ùî ôðàêö³ÿ êàëü ìî äóë³í-çâ’ÿ çó âàëü íèõ á³ëê³â
çäàò íà ôîñ ôî ðè ëþ âà òè ðå êîìá³íà íòí³ íàä ñï³ðàë³çî âà íó òà SH3
äî ìåí-âì³ñíó ä³ëÿí êè ITSN1 in vitro çà ïðè ñóò íîñò³ ³îí³â êàëüö³þ ³
êàëü ìî äóë³íó. Âèñ íîâ êè. Íàäñï³ðàë³çî âà íà òà SH3 äî ìåí-âì³ñíà
ä³ëÿí êè ITSN1 ï³äëÿ ãà þòü êàëüö³é/êàëü ìî äóë³í-çà ëåæ íî ìó ôîñ -
ôî ðè ëþ âàí íþ in vitro, ùî äîç âî ëÿº ïðè ïóñ òè òè ³ñíó âàí íÿ ðå ãó ëÿ-
ö³¿ ITSN1 êàëüö³ºâîþ ñèã íàëü íîþ ñèñ òå ìîþ.
Êëþ ÷îâ³ ñëî âà: ITSN1, ôîñ ôî ðè ëþ âàí íÿ, êàëü ìî äóë³í, êàëü-
ö³ºâà ñèã íàë³çàö³ÿ.
Êàëü öèé/êàëü ìî äó ëèí-çà âè ñè ìîå ôîñ ôî ðè ëè ðî âà íèå
ýí äî öè òîç íî ãî àäàï òî ðà ITSN1
Ä. Å. Ìîð äå ðåð, À. Â. Íè êî ëà åí êî, È. ß. Ñêðèï êè íà,
Î. Â. Ðû ìà ðåí êî, Ñ. Â. Êðî ïèâ êî, Ë. À. Öûáà, À. Â. Ðûí äè÷
ITSN1 – ýòî ýí äî öè òîç íûé àäàï òîð íûé áå ëîê, âû ïîë íÿ þ ùèé çíà -
÷è òåëü íûå ôóíê öèè â ñè íàï òè ÷åñ êîé ïå ðå äà ÷å íå ðâíî ãî èì ïóëü -
ñà. Èçâåñ òíî, ÷òî êàëü öè å âàÿ ñèã íà ëè çà öèÿ ÿâ ëÿ åò ñÿ êëþ ÷å âûì
ýëå ìåí òîì ðå ãó ëÿ öèè ôóíê öè î íè ðî âà íèÿ ñè íàï òè ÷åñ êèõ áåë êîâ.
Öåëü. Èññëå äî âàòü âîç ìîæ íîñòü êàëü öèé/êàëü ìî äó ëèí-çà âè ñè ìî-
ãî ôîñ ôî ðè ëè ðî âà íèÿ ITSN1. Ìå òî äû. Àôôèííàÿ õðî ìà òîã ðà ôèÿ,
êè íàç íàÿ ðå àê öèÿ in vitro, Âåñ òåðí áëîò-ãèá ðè äè çà öèÿ, îêðà øè âà -
íèå áåë êîâ ôëó î ðåñ öåí òíû ìè êðà ñè òå ëÿ ìè. Ðå çóëü òà òû. Ïî êà çà-
íî, ÷òî ôðàê öèÿ êàëü ìî äó ëèí-ñâÿ çû âà þ ùèõ áåë êîâ ñïî ñîá íà ôîñ -
ôî ðè ëè ðî âàòü ðå êîì áè íàí òíûå ñó ïåðñïè ðà ëè çî âàí íûé è SH3 äî -
ìåí-ñî äåð æà ùèé ó÷àñ òêè ITSN1 in vitro â ïðè ñó òñòâèè èî íîâ
êàëü öèÿ è êàëü ìî äó ëè íà. Âû âî äû. Có ïåð ñïè ðà ëè çî âàí íûé è SH3
äî ìåí-ñî äåð æà ùèé ó÷àñ òêè ITSN1 ïîä ëå æàò êàëü öèé/êàëü ìî äó -
ëèí-çà âè ñè ìî ìó ôîñ ôî ðè ëè ðî âà íèþ in vitro, ÷òî ïî çâî ëÿ åò ïðåä -
ïî ëî æèòü ñó ùåñ òâî âà íèå ðå ãó ëÿ öèè ITSN1 êàëü öè å âîé ñèã íàëü-
íîé ñèñ òå ìîé.
Êëþ÷åâûå ñëî âà: ITSN1, ôîñ ôî ðè ëè ðî âà íèå, êàëü ìî äó ëèí, êàëü-
öèå âàÿ ñèã íà ëè çà öèÿ.
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MORDERER D. YE. ET AL.
WB:
Anti-STOP
Sypro RUBY
staining
A
B
GST-SH3A-E
GST-CCR
GST
ATP
Calmodulin-binding
proteins
Ca/calmodulin
EGTA
Pro-Q
Diamond
staining
1 2 3 4
¬ STOP
Fig. 2. CCR and SH3A-E regions of ITSN1 are phosphorylated in vitro
by calmodulin-binding kinases in the presence of Ca/calmodulin: A –
calmodulin-binding proteins were isolated from mouse brain lysate by
affinity chromatography on calmodulin-agarose (bound proteins were
visualized by Sypro RUBY staining, efficiency of purification was che-
cked by the presence of STOP protein in the sample: 1 – brain lysate, 2 –
flowthrough, 3 – wash, 4 – elution of bound proteins); B – recombinant
GST-fused fragments of ITSN1 were used as substrates for in vitro ki-
nase reaction with calmodulin-binding proteins. Phosphorylated pro-
teins were visualized by Pro-Q Diamond staining
|