Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry
ITSN1 is a scaffold protein involved in endocytosis, signal transduction and cytoskeleton regulation. It has been previously shown that ITSN1 undergoes Ca²⁺/calmodulin-dependent phosphorylation in vitro. Aim. We intend to identify these phosphorylation sites. Methods. In vitro kinase reaction; liqui...
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Інститут молекулярної біології і генетики НАН України
2015
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| Цитувати: | Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry / D.Ye. Morderer, O.V. Nikolaienko, A.V. Rynditch // Вiopolymers and Cell. — 2015. — Т. 31, № 5. — С. 338-344. — Бібліогр.: 28 назв. — англ. |
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irk-123456789-1525922019-06-13T01:27:29Z Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry Morderer, D.Ye. Nikolaienko, O.V. Rynditch, A.V. Structure and Function of Biopolymers ITSN1 is a scaffold protein involved in endocytosis, signal transduction and cytoskeleton regulation. It has been previously shown that ITSN1 undergoes Ca²⁺/calmodulin-dependent phosphorylation in vitro. Aim. We intend to identify these phosphorylation sites. Methods. In vitro kinase reaction; liquid chromatography-tandem mass spectrometry (LC/MS/MS). Results. We identified five sites of Ca²⁺/calmodulin-dependent phosphorylation in the recombinant fragments of ITSN1. Conclusions. We have shown that the ITSN1 coiled-coil region (CCR) and the interdomain linkers between EH2 and CCR, SH3A and SH3B, SH3B and SH3C domains were phosphorylated in a Ca²⁺/calmodulin-dependent manner in vitro. ITSN1 – це скафолдний білок, задіяний у процесах ендоцитозу, сигнальної трансдукції та регуляції цитоскелету. Раніше було показано, що ITSN1 підлягає Ca²⁺/кальмодулін-залежному фосфорилюванню in vitro. Мета. Ідентифікувати сайти цього фосфорилювання. Методи. In vitro кіназна реакція; рідинна хроматографія, поєднана з тандемною мас-спектрометрією (LC/MS/MS). Результати. Ми ідентифікували 5 сайтів Ca²⁺/кальмодулін-залежного фосфорилювання у рекомбінантних фрагментах ITSN1. Висновки. Було показано, що надспіралізована ділянка (CCR) та міждоменні лінкери між ЕН2 та CCR, SH3A та SH3B, а також між SH3B та SH3C доменами ITSN1 підлягають Ca²⁺/кальмодулін-залежному фосфорилюванню. ITSN1 является скаффолдным белком, задействованным в процессах эндоцитоза, сигнальной трансдукции и регуляции цитоскелета. Ранее было показано, что ITSN1 подлежит Ca²⁺/кальмодулин-зависимому фосфорилированию in vitro. Цель Идентифицировать сайты Ca²⁺/кальмодулин-зависимого фосфорилирования ITSN1. Методы. In vitro киназная реакция, жидкостная хроматография в сочетании с тандемной масс-спектрометрией (LC/MS/MS). Результаты. Мы идентифицировали 5 сайтов Ca²⁺/кальмодулин-зависимого фосфорилирования в рекомбинантных фрагментах ITSN1. Выводы. Было показано, что суперспирализированый участок (CCR) и междоменные линкеры между ЕН2 и CCR, SH3A и SH3B, а также между SH3B и SH3C доменами ITSN1 подвергаются Ca²⁺/кальмодулин-зависимому фосфорилированию. 2015 Article Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry / D.Ye. Morderer, O.V. Nikolaienko, A.V. Rynditch // Вiopolymers and Cell. — 2015. — Т. 31, № 5. — С. 338-344. — Бібліогр.: 28 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008F5 http://dspace.nbuv.gov.ua/handle/123456789/152592 577.29 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| topic |
Structure and Function of Biopolymers Structure and Function of Biopolymers |
| spellingShingle |
Structure and Function of Biopolymers Structure and Function of Biopolymers Morderer, D.Ye. Nikolaienko, O.V. Rynditch, A.V. Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry Вiopolymers and Cell |
| description |
ITSN1 is a scaffold protein involved in endocytosis, signal transduction and cytoskeleton regulation. It has been previously shown that ITSN1 undergoes Ca²⁺/calmodulin-dependent phosphorylation in vitro. Aim. We intend to identify these phosphorylation sites. Methods. In vitro kinase reaction; liquid chromatography-tandem mass spectrometry (LC/MS/MS). Results. We identified five sites of Ca²⁺/calmodulin-dependent phosphorylation in the recombinant fragments of ITSN1. Conclusions. We have shown that the ITSN1 coiled-coil region (CCR) and the interdomain linkers between EH2 and CCR, SH3A and SH3B, SH3B and SH3C domains were phosphorylated in a Ca²⁺/calmodulin-dependent manner in vitro. |
| format |
Article |
| author |
Morderer, D.Ye. Nikolaienko, O.V. Rynditch, A.V. |
| author_facet |
Morderer, D.Ye. Nikolaienko, O.V. Rynditch, A.V. |
| author_sort |
Morderer, D.Ye. |
| title |
Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry |
| title_short |
Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry |
| title_full |
Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry |
| title_fullStr |
Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry |
| title_full_unstemmed |
Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry |
| title_sort |
identification of ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold itsn1 by tandem mass spectrometry |
| publisher |
Інститут молекулярної біології і генетики НАН України |
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2015 |
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Structure and Function of Biopolymers |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/152592 |
| citation_txt |
Identification of Ca²⁺/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry / D.Ye. Morderer, O.V. Nikolaienko, A.V. Rynditch // Вiopolymers and Cell. — 2015. — Т. 31, № 5. — С. 338-344. — Бібліогр.: 28 назв. — англ. |
| series |
Вiopolymers and Cell |
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2025-07-14T04:03:15Z |
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| fulltext |
338
D. Ye. Morderer, O. V. Nikolaienko, A. V. Rynditch
© 2015 D. Ye. Morderer et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
Structure and Function
of Biopolymers
ISSN 0233-7657
Biopolymers and Cell. 2015. Vol. 31. N 5. P. 338–344
doi: http://dx.doi.org/10.7124/bc.0008F5
UDC 577.29
Identification of Ca2+/calmodulin-dependent phosphorylation sites
of endocytic scaffold ITSN1 by tandem mass spectrometry
D. Ye. Morderer1, O. V. Nikolaienko2, A. V. Rynditch1
1Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2 Department of Biomedicine, University of Bergen,
Jonas Lies vei 91, Bergen, Norway, 5009
d.y.morderer@imbg.org.ua
ITSN1 is a scaffold protein involved in endocytosis, signal transduction and cytoskeleton regulation. It has
been previously shown that ITSN1 undergoes Ca2+/calmodulin-dependent phosphorylation in vitro. Aim. We
intend to identify these phosphorylation sites. Methods. In vitro kinase reaction; liquid chromatography-tan-
dem mass spectrometry (LC/MS/MS). Results. We identified five sites of Ca2+/calmodulin-dependent phos-
phorylation in the recombinant fragments of ITSN1. Conclusions. We have shown that the ITSN1 coiled-coil
region (CCR) and the interdomain linkers between EH2 and CCR, SH3A and SH3B, SH3B and SH3C do-
mains were phosphorylated in a Ca2+/calmodulin-dependent manner in vitro.
K e y w o r d s: ITSN1, Ca2+, phosphorylation, LC/MS/MS.
Introduction
ITSN1 is a scaffold protein implicated in various
cellular processes including the endocytosis, signal
propagation through a number of signaling path-
ways, actin cytoskeleton regulation, etc [1].
Accumulating evidence connects ITSN1 misregula-
tion with the neurodevelopmental and neurodegen-
erative disorders, such as Down syndrome,
Alzheimer’s disease and Huntington’s disease [2–4].
It is believed that ITSN1 operates in a cell by inter-
acting with the partner molecules, thereby promot-
ing an assembly of macromolecular complexes. As
for today, dozens of ITSN1 interactors are already
known and their number continues to increase [5].
Such complexity implies the existence of precise
regulatory mechanisms which control the dynamics
of interaction and the selection of partner molecules
for ITSN1 binding. The post-translational modifica-
tions of ITSN1 can potentially be involved in these
mechanisms.
Phosphorylation is one of the most widespread and
well studied post-translational modifications of pro-
teins. It results in the covalent attachment of phos-
phate group to the serine, threonine or tyrosine resi-
dues, which can lead to an alteration in the protein
conformation and subsequent change of its properties.
Therefore, phosphorylation is a common mechanism
for triggering the protein activation state. ITSN1
phosphorylation has been already revealed in several
large-scale studies of phosphoproteome in different
tissues [6–9]. Additionally, the tyrosine phosphoryla-
tion of ITSN1 in response to the overexpression of
Epstein-Barr virus protein LMP2A and tyrosine ki-
nase Syk in HEK293 cells has been reported [10].
However, it is unclear which intracellular pathways
339
Identification of Ca2+/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry
drive these phosphorylation events. Recently, we have
demonstrated that the CCR and SH3A-E fragments of
ITSN1, containing the coiled-coil region and SH3 do-
mains, respectively (Fig. 1A), can be phosphorylated
in the Ca2+/calmodulin-dependent manner, suggesting
the possibility of ITSN1 phosphorylation in response
to the elevation of the intracellular Ca2+ concentration
[11]. Here we report the identification of Ca2+/calmod-
ulin-dependent phosphorylation sites in ITSN1 using
tandem mass spectrometry combined with liquid
chromatography (LC/MS/MS).
Materials and Methods
Plasmid constructions
The plasmid encoding GST-fused CCR fragment
of ITSN1 was described previously [12]. Nucleotide
sequence encoding SH3A-E fragment of ITSN1 was
PCR amplified and cloned in pGEX-4T-2 vector (GE
Healthcare, USA).
Recombinant protein expression and purification.
Recombinant GST-fused proteins were expressed
using Escherichia coli BL21(DE3) strain. Obtained
proteins were affinity-purified using glutathione-
sepharose 4B (GE Healthcare, USA) according to
manufacturer’s instruction.
In vitro kinase reaction
The reaction was performed as described previously
[11]. Briefly, calmodulin-binding proteins were puri-
fied from mouse brain lysate on calmodulin-agarose
beads (Sigma-Aldrich, USA), dialyzed against kinase
buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mM
DTT) and then mixed with purified recombinant ITSN1
fragments. The reaction was supplied by 0.4 mM
EGTA, 0.5 µM microcystin, 100 µM ATP, 3 µM
calmodulin, 2 mM CaCl2, and incubated at 30 ̊ C for 30
min. Then the reaction was stopped by addition of an
equal volume of Laemmli sample buffer (150 mM
Tris–HCl pH 6.8, 2.5 % glycerol, 10% SDS, 3 %
β-mercaptoethanol and 0.5 % bromophenol blue). After
boiling the samples were resolved by SDS-PAGE and
stained by Coomassie Brilliant Blue.
LC/MS/MS
In-gel digestion of proteins was performed at 37°C
overnight with trypsin or Glu-C proteases (Roche,
Switzerland) (1:50 protease : protein ratio). Next day
the peptides were extracted from the gel using Oasis
C18 kit. Then, the peptides were dissolved to a final
concentration of 4 % in formic acid before analysis
on LTQ Orbitrap Velos Pro mass spectrometer
(Thermo Fischer Scientific, USA) equipped with a
Fig. 1. A – Schematic repre-
sentation of ITSN1 domain
structure. Domains studied in
this work are underlined. B –
Aminoacid sequences of stud-
ied fragments of ITSN1. Pep-
tides identified by mass spec-
trometry are enlarged and un-
derlined. Peptides identified
after treatment with trypsin
are highlighted in bold. Pep-
tides identified after treatment
with Glu-C are in italics
A
B
340
D. Ye. Morderer, O. V. Nikolaienko, A. V. Rynditch
nano spray Flex ion source (Thermo Fischer
Scientific, USA), coupled to a Dionex Ultimate NCS-
3000 LC system (Thermo Fischer Scientific, USA).
Fragmentation of primary ions was performed by
HCD (high-energy collision dissociation) technique.
Tandem mass spectra were extracted by Proteome
DiscovererTM software (Thermo Scientific, USA).
Charge state deconvolution and deisotoping were not
performed. All MS/MS samples were analyzed using
Mascot (Matrix Science, London, UK; version
1.3.0.339). Mascot was set up to search Mascot5_
SwissProt_Homo sapiens (human) (16369 entries)
assuming the digestion enzyme trypsin or V8. Mascot
was searched with a fragment ion mass tolerance of
0.05 Da and a parent ion tolerance of 15 PPM.
Iodoacetamide derivative of cysteine was specified in
Mascot as a fixed modification. Oxidation of methio-
nine, acetylation of the N-terminus and phosphoryla-
tion of serine and threonine were specified in Mascot
as variable modifications. The phosphopeptides with
reliably identified phosphorylation site were selected
by the value of Mascot Delta Ion Score, using value
13 as a threshold [13]. Their spectra were also manu-
ally revised using Scaffold software version 3.6.5
(Proteome Software) [14].
Results and Discussion
In order to identify the sites of Ca2+/calmodulin-
dependent phosphorylation in the ITSN1 recombi-
nant fragments we repeated our previously described
assay for in vitro Ca2+/calmodulin-dependent phos-
phorylation [11] and subsequently analyzed the sam-
ples by LC/MS/MS. To increase the sequence cover-
age and probability of identification for all possible
phosphosites, the samples were divided and digested
independently by two distinct endoproteases: trypsin
and Glu-C. As a result of the mass spectrometric
peptide detection we obtained 48.7 % coverage for
CCR sequence and 59.4 % coverage for SH3A-E se-
quence using trypsin, whereas for Glu-C the corre-
sponding values were 39.1 % and 23.3 %, respec-
tively. In summary we obtained 61.6 % and 67.4 %
sequence coverage for CCR and SH3A-E fragments,
respectively (Fig.1B).
For each digestion variant except the SH3A-E frag-
ment digested by Glu-C we were able to identify a
number of phosphopeptides (Supplementary Table 1).
The obtained phosphopeptides were sorted according
to their calculated Mascot Delta Ion Score value to se-
lect those with the most confidently located phosphor-
ylation sites. In this way we identified five phospho-
peptides containing the phosphorylation sites corre-
sponding to positions T349, T567, S624, S904 and
S978 in ITSN1 sequence (Q15811 in UniProtKB data-
base) (Table 1). We also carefully checked the MS/MS
spectra of selected peptides manually for the presence
of phosphospecific secondary ions to confirm a reli-
able location of the phosphorylated sites (Fig. 2).
Table 1. Identification of Phosphorylation Sites in ITSN1 Fragments by LC/MS/MS
Peptide Sequence
Po
si
tio
n
of
Ph
os
ph
os
it-
ei
n
IT
SN
1
(Q
15
81
1)
Fr
ag
m
en
t
Protease
Used Pe
pt
id
e
Id
en
tifi
ca
tio
n
Pr
ob
ab
ili
ty
M
as
co
t I
on
Sc
or
e Mascot
Identity
Score
Mascot Delta
Ion Score
M
od
ifi
ca
tio
ns
(R)DSLVtLKR(A) 567 CCR Trypsin 95 % 57.93 28.5248 19.68 Phospho
(+80)
(K)KLPVtFEDK(K) 349 CCR Trypsin 95 % 35.35 25.0 35.35 Phospho
(+80)
(E)IHNKQQLQKQKsMEAERLKQKE(Q) 624 CCR Glu-C 95 % 35.18 26.374897 35.18 Phospho
(+80)
(R)SAFTPATATGSSPsPVLGQGEK(V) 904 SH3A-E Trypsin 95 % 86.77 25.0 15.0099945 Phospho
(+80)
(K)STsMDSGSSESPASLKR(V) 978 SH3A-E Trypsin 95 % 117.05 29.604706 16.410004 Phospho
(+80)
341
Identification of Ca2+/calmodulin-dependent phosphorylation sites of endocytic scaffold ITSN1 by tandem mass spectrometry
Fig. 2. HCD MS/MS spectra of identified
phosphopeptides. A – HCD spectrum of (K)
KLPVpTFEDK(K) (residues 345–353). The
most intensive peak corresponds to the neu-
tral loss of H3PO4 from parental ion (–98),
indicating phosphorylation of the peptide.
B – HCD spectrum of (R)DSLVpTLKR(A)
(residues 563–570). Phosphorylation of
T567 is confirmed by the presence of y4, y5
and y6 ions with the neutral losses of H3PO4
(–98). C – HCD spectrum of (E)IHNK QQ-
LQ KQK pSME A ER LK QKE(Q) (residues
613–634). Phosphorylation of S624 is indi-
cated by y11 and y12 ions with the neutral
losses of H3PO4 (–98). D – HCD spectrum
of (R)SAFTPA TATG SS PpSPVLGQGEK(V)
(residues 891–912). Phosphorylation of the
peptide is indicated by parental ion with the
neutral loss of H3PO4 (-98). Assignment of
phosphorylation to S904 site is justified by
the presence of y10 ion. E – HCD spectrum
of (K)STp SMD SGS SES PASLKR(V) (resi-
dues 976–992). Phosphorylation of S978 [is]
indicated by b2 and b3 ions, as well as by the
presence of b3 ions containing losses of
H3PO4 (-98) and H2O
+ H3PO4 (–116)
Fig. 3. A – Location of phos-
phorylation sites within the
fragments of ITSN1. B – Par-
tial sequence of SH3A-E be-
tween SH3A and SH3C do-
mains. Domain sequences are
shown in grey. Serine/threo-
nine residues within domains
are shown in blue and within
interdomain linkers – in red.
Frequency of occurrence of
serine/threonine residues in
the interdomain linkers is
higher than inside domains
A
B
A
B
C
D
E
342
D. Ye. Morderer, O. V. Nikolaienko, A. V. Rynditch
Among the identified phosphorylation sites, only
S624 and S978 match the R/K-X-X-S/T canonical
recognition motif for Ca2+/calmodulin-dependent ki-
nases [15]. Amino acid sequences of other sites do
not resemble the known kinase recognition motifs,
thus making a kinase responsible for these phos-
phorylation events difficult to predict. However, it is
known that the absence of full match with the con-
sensus motif does not mean the inability of a particu-
lar kinase to phosphorylate a given substrate. For
instance, it has been shown that cyclin-dependent
linase CDK1 under certain condition can phosphory-
late the minimal S/T-P recognition motif instead of
the canonical S/T-P-X-K/R motif [16, 17].
Interestingly, the identified S904 site corresponds to
this minimal motif. Additionally, the S904 phos-
phorylation site is flanked by proline residues, indi-
cating its possible phosphorylation by so called pro-
line-directed kinases, which include the CDK,
MAPK, JNK and GSK kinase families [18].
Interestingly, CDK4 and CDK5 can interact with
calmodulin and be activated by either Ca2+ or Ca2+-
dependent phosphorylation [19–22], suggesting the
possibility of their presence in our purified calmodu-
lin-binding fraction and the activation in Ca2+-
dependent manner. Notably, the S904 phosphoryla-
tion has already been detected in the large-scale
studies of phosphoproteome [6–9].
For S349 and T567 it is hard to predict a kinase
that could phosphorylate these residues. Interestingly,
both of them are surrounded by hydrophobic amino
acids (at positions –2, –1 and +1), suggesting their
modification by the same kinase.
A functional role of the identified phosphorylation
sites is unclear and needs to be studied in future ex-
periments. The T567 and S624 sites are located in the
coiled-coil region of ITSN1 (Fig.3A). This domain
mediates its oligomerization or interactions with other
proteins containing similar domains [23]. It has been
shown that phosphorylation of the threonine residues
within such regions can destabilize helices, whereas
phosphorylation of the serine residues can lead to ei-
ther their stabilization or destabilization [24, 25]. It
can be expected that the phosphorylation events with-
in the coiled-coil region can affect its ability to oligo-
merize or interact with other proteins. T349, S904 and
S904 are located in the interdomain linkers that are
predicted to be intrinsically disordered (Fig. 3A). It
has been demonstrated that phosphorylation of the
serine and threonine residues most often occurs in
such regions [26–28]. Moreover, the S904 and S978
sites are located within the regions enriched in the ser-
ine/threonine residues (Fig.3B). Phoshorylation of
several residues in these clusters has been identified
[6–8]. Interestingly, these two clusters are located at
both sides and in close proximity to the SH3B do-
main. Therefore, we suggest that phosphorylation of
the serine and threonine residues belonging to these
clusters can affect the SH3B domain ability to medi-
ate protein-protein interactions. We believe that ex-
amination of the role of identified phosphorylation
sites in the ITSN1 function is a promising direction
for further investigations.
Conclusions
We showed that ITSN1 coiled-coil region (CCR)
and interdomain linkers between the EH2 and CCR,
SH3A and SH3B, SH3B and SH3C domains were
phosphorylated in Ca2+/calmodulin-dependent man-
ner in vitro.
Funding.
This work was supported by joint project between
NAS of Ukraine and CNRS “From Molecular to
Cellular Events in Human Pathologies”
(№0113U002831).
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Ідентифікація сайтів Са2+/кальмодулін-залежного
фосфорилювання скаффолдного білка ендоцитозу
ITSN1 за допомогою тандемної мас-спектрометрії.
Д. Є. Мордерер, О. В. Ніколаєнко, А. В. Риндич.
ITSN1 – це скафолдний білок, задіяний у процесах ендоцитозу,
сигнальної трансдукції та регуляції цитоскелету. Раніше було
показано, що ITSN1 підлягає Са2+/кальмодулін-залежному
фосфорилюванню in vitro. Мета Ідентифікувати сайти цього
фосфорилювання. Методи. In vitro кіназна реакція; рідинна
хроматографія, поєднана з тандемною мас-спектрометрією
(LC/MS/MS). Результати. Ми ідентифікували 5 сайтів Са2+/
кальмодулін-залежного фосфорилювання у рекомбінантних
фрагментах ITSN1. Висновки. Було показано, що надспіралі-
зована ділянка (CCR) та міждоменні лінкери між ЕН2 та CCR,
344
D. Ye. Morderer, O. V. Nikolaienko, A. V. Rynditch
SH3A та SH3B, а також між SH3B та SH3C доменами ITSN1
підлягають Са2+/кальмодулін-залежному фосфорилюванню.
К л юч ов і с л ов а: ITSN1, Ca2+, фосфорилювання, LC/MS/
MS.
Идентификация сайтов Са2+/кальмодулин-зависимого
фосфорилирования скаффолдного белка эндоцитоза
ITSN1.
Д. Е. Мордерер, А. В. Николаенко, А. В. Рындич.
ITSN1 является скаффолдным белком, задействованным в про-
цессах эндоцитоза, сигнальной трансдукции и регуляции ци-
тоскелета. Ранее было показано, что ITSN1 подлежит Са2+/
кальмодулин-зависимому фосфорилированию in vitro. Цель
Идентифицировать сайты Са2+/кальмодулин-зависимого фос-
форилирования ITSN1. Методы. In vitro киназная реакция,
жидкостная хроматография в сочетании с тандемной масс-
спектрометрией (LC/MS/MS). Результаты. Мы идентифици-
ровали 5 сайтов Са2+/кальмодулин-зависимого фосфорилиро-
вания в рекомбинантных фрагментах ITSN1. Выводы. Было
показано, что суперспирализированый участок (CCR) и
междоменные линкеры между ЕН2 и CCR, SH3A и SH3B, а
также между SH3B и SH3C доменами ITSN1 подвергаются
Са2+/кальмодулин-зависимому фосфорилированию.
К л юч е в ы е с л ов а: ITSN1, Ca2+, фосфорилирование, LC/
MS/MS.
Received 03.07.2015
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