Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro
Coenzyme A-synthases α and β (CoASy α and CoASy β ) contain proline rich regions which may bring them into complexes with SH3-domain containing proteins. To test whether CoASy isoforms can bind to SH3 domains we performed in vitro pull down experiments. It was found that CoASy β N-terminal extension...
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| Zitieren: | Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro / O.S. Breus, G.G. Panasyuk, I.T. Gout, V.V. Filonenko, I.O. Nemazanyy // Біополімери і клітина. — 2008. — Т. 24, № 2. — С. 123-128. — Бібліогр.: 16 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859464728635506688 |
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| author | Breus, O.S. Panasyuk, G.G. Gout, I.T. Filonenko, V.V. Nemazanyy, I.O. |
| author_facet | Breus, O.S. Panasyuk, G.G. Gout, I.T. Filonenko, V.V. Nemazanyy, I.O. |
| citation_txt | Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro / O.S. Breus, G.G. Panasyuk, I.T. Gout, V.V. Filonenko, I.O. Nemazanyy // Біополімери і клітина. — 2008. — Т. 24, № 2. — С. 123-128. — Бібліогр.: 16 назв. — англ. |
| collection | DSpace DC |
| container_title | Біополімери і клітина |
| description | Coenzyme A-synthases α and β (CoASy α and CoASy β ) contain proline rich regions which may bring them into complexes with SH3-domain containing proteins. To test whether CoASy isoforms can bind to SH3 domains we performed in vitro pull down experiments. It was found that CoASy β N-terminal extension, which is especially abundant in prolines, can interact specifically and directly with SH3 domains of tyrosine kinases Fyn and CSK, phospholipase Cγ, NADPH oxidase activator 1 – p67phox, and cytoskeleton protein spectrin. Furthermore, C-terminal SH3 domain of p67phox can also interact with SH3 binding site that resides on the shared part of CoASyβ and CoASyα . These data demonstrated that CoA Synthases could be involved in complexes with signaling proteins in living cells which may regulate enzymatic activities of CoA Synthases or vice versa CoA Synthase may modulate some steps in signal transduction in the cell in currently unknown way.
Кофермент А-синтази α та β містять збагачені проліном ділянки, які можуть сприяти їхньому комплексоутворенню з білками, що містять SH3 домени. Для перевірки цієї гіпотези ми провели експерименти in vitro по зв’язуванню повнорозмірного білка КоА-синтази α та специфічного для КоА-синтази β N-кінцевого пептиду із SH3 доменами сигнальних білків. Виявилося, що специфічний для КоА-синтази β N-кінцевий пептид, багатий на пролін, специфічно взаємодіє з SH3 доменами тирозинових протеїнкіназ Fyn і CSK, фосфоліпази Cγ , активатора NADPH оксидази 1 – p67phox і цитоскелетного білка спектрину. Крім того, SH3 домен p67phox зв’язується із збагаченою проліном послідовністю, спільною для обох ізоформ. Отримані результати дозволяють припустити, що КоА-cинтази α і β перебувають у комплексах з сигнальними білками і, таким чином, відбувається регуляція їхньої ферментативної активності або ж КоА-синтази невідомим на даний час способом модулюють певні етапи передавання сигналів у клітині.
Кофермент А-синтазы α и β содержат богатые пролином области, возможно, способствующие их комплексообразованию с белками, содержащими SH3 домены. Для проверки этой гипотезы мы провели эксперименты по связыванию in vitro полноразмерного белка КоА-синтазы α и специфического для КоА-синтазы β N-концевого пептида с SH3 доменами сигнальных белков. Оказалось, что пролин-богатый N-концевой пептид КоА-синтазы βспецифически взаимодействует с SH3 доменами тирозиновых протеинкиназ Fyn и CSK, фосфолипазы Cγ, активатора NADPH оксидазы 1 – p67phox и белка цитоскелета спектрина. Кроме этого, SH3 домен p67phox взаимодействует с обогащенной пролином последовательностью, общей для обеих изоформ. Полученные результаты позволяют предположить, что КоА-синтазы α и β образуют комплексы с сигнальными белками и, таким образом, регулируется ферментативная активность КоА-синтаз либо же КоА-синтазы неизвестным на данный момент способом модулируют определенные этапы передачи сигналов в клетке.
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| first_indexed | 2025-11-24T04:44:46Z |
| format | Article |
| fulltext |
Proline rich regions of coenzyme A synthase α and β
interact with SH3 domains of signaling proteins in vitro
O. S. Breus1, G. G. Panasyuk1,2, I. T. Gout2, V. V. Filonenko1, I. O. Nemazanyy1
1Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
150 Zabolotnogo Str., Kyiv 03680, Ukraine
2Department of Biochemistry and Molecular Biology, University College London
Gower Street, London WC1 E6BT, UK
filonenko@imbg.org.ua
Сoenzyme A-synthases α and β (CoASy α and CoASy β) contain proline rich regions which may bring them
into complexes with SH3-domain containing proteins. To test whether CoASy isoforms can bind to SH3
domains we performed in vitro pull down experiments. It was found that CoASy β N-terminal extension,
which is especially abundant in prоlines, can interact specifically and directly with SH3 domains of tyrosine
kinases Fyn and CSK, phospholipase Cγ, NADPH oxidase activator 1 – p67phox, and cytoskeleton protein
spectrin. Furthermore, C-terminal SH3 domain of p67phox can also interact with SH3 binding site that
resides on the shared part of CoASy β and CoASy α. These data demonstrated that CoA Synthases could be
involved in complexes with signaling proteins in living cells which may regulate enzymatic activities of CoA
Synthases or vice versa CoA Synthase may modulate some steps in signal transduction in the cell in
currently unknown way.
Key words: CoA Synthase, SH3 domain, proline rich regions, signaling proteins.
Introduction. Coenzyme A (СoA) is ubiquitous
cofactor essential for living, involved in numerous
enzymatic reactions in cell. Reduced CoA and its acyl-
and acetyl-derivatives play a key role in the energetic,
carbohydrate, and lipid cell metabolism [1], regulation
of transcription via histone acetylation [2], proteins
acylation [3], and oxidative stress protection via
cysteamine biosynthesis [1]. Both quantity and form of
available CoA and its derivatives depend on cell type
and cell compartment, and vary in different
physiological circumstances [1].
It has been shown that the level of intracellular CoA
is regulated by various extracellular stimuli including
insulin, glucose, fatty acids, pyruvate, glucagon, and
glucocorticoids [1, 6–8]. The rate of CoA biosynthesis
changes with fasting, re-feeding, and several
pathological conditions, such as diabetes, Reye
syndrome, and cancer [1, 6–8].
However, very little is known about the pathways
leading to such regulation. Up-to-date, the mechanisms
of regulation are known only for the first enzymatic
step in the pathway of CoA biosynthesis –
phosphorylation of pantothenate by pantothenate
kinase (PanK). There are at least four PANK genes in
human genome (hPANK1, hPANK2, hPANK3, and
hPANK4) and at least two alternative splicing isoforms
for PanK1 and PanK2. PanKs share the same
catalytical core but are differently regulated by reduced
CoA and its thioesters such as acetyl-CoA and
acyl-CoA [4, 9–11]. Differential transcription of
123
ISSN 0233-7657. Біополімери і клітина. 2008. Т. 24. № 2 doi 10.7124/bc.000799
O. S. BREUS, G. G. PANASYUK, I. T. GOUT, V. V. FILONENKO,
I. O. NEMAZANYY, 2008
PANKs mRNA in response to different stimuli such as
hypolipidemic agents, glucose, and lipids was also
shown to modulate intracellular CoA level [10].
4'-phosphopantetheine is one of the major
pantothenate metabolites in cell along with CoA and
pantothenate itself. It means that CoA Synthase
(CoASy), which utilizes 4'-phosphopantetheine to
generate dephosphoCoA and CoA, may be the second
rate limiting enzyme in the pathway of CoA
biosynthesis [4]. Nevertheless, no physiological
regulators of CoASy have been reported so far.
Recently two isoforms of CoA Synthase – CoASy α
and CoASy β – have been cloned [13, 15]. We have
demonstrated that CoASy α associates specifically
with ribosomal S6 kinase – one of the key regulators of
cell size and growth. The association was observed
between native and transiently overexpressed proteins
in vivo [15]. New interactions with signaling molecules
could uncover novel mechanisms of CoA biosynthesis
regulation.
The distinctive feature of CoASy β is a proline-rich
29 amino acids (29 aa) long N-terminal extension (Fig.
1, A). According to bioinformatic analysis this
proline-rich sequence may bring CoASy β into different
signaling complexes with Src homology 3 (SH3)
domain containing proteins in cell. SH3 domains are
small modular domains about 60 amino acids in size.
They function as adapters, mediating protein-protein
interactions, and are common domains for proteins
building signaling networks and for some cytoskeleton
proteins. SH3 domains are found in a wide range of
functionally distinct signaling proteins including
adapter proteins, protein kinases, lipid kinases,
GTPases, GAPs, GEFs, lipases, protein phosphatases,
etc. These domains bind with moderate affinity and
selectivity to proline-rich ligands and play critical roles
in numerous biological processes ranging from
regulation of enzymes by intramolecular interactions,
increasing the local concentration or altering the
subcellular localization of components of signaling
pathways, and mediating the assembly of large
multiprotein complexes [5].
In this paper, we show that full-length CoA
Synthase α and N-terminal 29 aa extension of CoASy β
isoform can directly interact with SH3 domains of a
number of signaling proteins in vitro.
Material and Methods. Bioinformatics. Scansite
program was used for bioinformatical identification of
protein motives, which could potentially bind to
domains such as SH2 and/or SH3. Scansite search is
based on the matrix of selectivity values for amino
acids at each position relative to an orienting residue as
determined by the oriented peptide library technique
[11].
Plasmid Construction and Expression studies. The
human EST clone 5187222, corresponding to the full
length CoASy β, was received from I.M.A.G.E.
consortium and verified by sequencing. The coding
sequence corresponding to the first N-terminal 29 aa of
CoASy β was amplified by PCR and cloned into
pET42a vector («Novagen», USA) in frame with GST
and 6HIS (29 aa CoASy β pET42a). 29 aa CoASy
β-pET42a were expressed in Escherichia coli BL21
DE3 cells and recombinant protein was then purified
using Ni-NTA agarose according to Ni-NTA Spin
Handbook manual («Qiagen», USA).
Pull Down Experiments. In a pull-down assay, 1 µg
of 6His-GST, 6His-GST-29 aa CoASy β fusion protein
and His-CoASy α protein were incubated with Ni-NTA
(«Qiagen») for 2 h at 4 °C in a buffer for native
condition (50 mM NaH2PO4, pH 8.0, 300 mM NaCl).
The beads were then washed with the binding buffer to
remove unbound proteins and incubated at 4 °C for 3 h
with 1 µg of purified GST-SH3 domains [16].
Non-specific interactions were removed by washing
with binding buffer and bound proteins were eluted by
boiling in Laemmli sample buffer.
Immunoblot Analysis. Proteins were separated by
SDS-PAGE and transferred onto polyvinylidene
difluoride membrane. After that the membrane was
incubated for 1 h in blocking solution (5 % non-skim
dry milk in TBS/Tween 0.1 %). After blocking the
membranes were cut on three pieces according to
molecular weight marker to visualize 62 kDa full length
CoASy α-6His protein with CoASy specific
antibodies, SH3 domains with molecular weights
ranging from 55 to 30 kDa with anti-GST antibodies or
28–25 kDa respectively 6His-GST-29 aa CoASy β, and
6His-GST proteins with anti-6His antibodies. After
washing with TBS, 0.1 % Tween, the membrane was
incubated for 1 h with secondary horseradish
peroxidase-conjugated anti-mouse antibody («Prome-
124
BREUS O. S. ET AL.
ga»). The antigen-antibody complexes were detected
using the ECL system («Amersham Biosciences»).
Results and Discussion. To elucidate specific
function of CoASy β isoform, which has 29 aa proline
rich extension at the N-terminus, we employed
bioinformatical approach. Using a medium stringency
Scansite [12] search, in both isoforms we revealed
potential binding sites for SH2/SH3 domains of
different proteins. Moreover, recognition motives
specific for SH3 domains of Crk, Abl, Nck2, PLCγ, Itk
proteins were identified only in 29 aa N-terminal
extension of the CoASy β, whereas SH3 domains of
cortactin, Grb2, Src, amphiphysin, Cbl-associated
protein and intersectin had predictive binding sites
residing on the CoASy β specific extension as well as
on the part of molecule, shared by β and α isoforms
(Fig. 1, B). These predictions are an important
indication of the possibility of CoASy involvement in
complexes with signaling proteins in living cells.
At the next stage we tested interactions of full
length CoASy α and CoASy β specific 29 aa extension
with SH3 domains of different intracellular proteins.
For this purpose, we cloned cDNA coding for 29 aa
fragment in frame with 5' sequence of GST-6His tag in
pET42a vector («Novagen»). Fusion with GST-6His
tags allowed 29 aa peptide to be expressed in bacteria
and purified on Ni-NTA agarose («Qiagen») (Fig. 2,
B). Purified protein was tested in Western blotting with
rabbit polyclonal antibodies generated against the
synthetic peptide composed from the first N-terminal
16 aa of CoASy β [13] (Fig. 1, C).
To examine direct interactions of 29 aa CoASy β
peptide and full-length CoASy α with SH3 domains,
we used a panel of 13 SH3 domains originated from
various mammalian proteins in pull down experiments.
This panel was generated as SH3-GST fusion proteins
expressed in E. coli. GST-6His-29 aa CoASy β purified
from bacteria and full-length 6His-CoASy α which was
purified earlier in our laboratory using baculovirus
system [13] were immobilized on Ni-NTA agarose.
SH3-GST domains were added to the resin coated with
the bait proteins, incubated for 2 h in binding buffer and
125
PROLINE RICH REGIONS COENZYME A SYNTHASE α AND β INTERACT WITH SH3
Fig. 1. Schematic representation of mammalian CoA Synthases: A –
domain organization of mammalian CoA Synthases α and β;
dPCoAK – dephosphoCoA kinase domain; PPAT – phospho-
pantetheine adenylyltransferase domain; N-domain – N-terminal
domains; amino acids are shown in the one letter code; B –
bioinformatic prediction of interactive sites on CoASy β N-terminal
29 aa extension. For prediction Scansite program was used
(http://scansite.mit.edu)
Fig. 2. Expression in E. coli and purification of GST-6His fused
N-terminal 29 aa extension of CoASy β: A – PAGE of total lysates of
BL-21 cells transformed with His-GST-29 aa CoASy β pET42a
before or after induction with 100 µM IPTG; Coomassie blue
staining; B – PAGE of His-GST-29 aa CoASy β fractions eluted
from Ni-NTA agarose with different imidazole concentrations;
Coomassie blue staining; C – Western blot analysis of purified
6His-GST-29 aa CoASy β protein with rabbit polyclonal antibodies
to N-terminal peptide of CoASy β
126
BREUS O. S. ET AL.
Fig. 3. In vitro binding of CoASy α and 29 aa
N-terminal extension of CoASy β with SH3 domains
of different proteins. The top panel, visualized with
anti-GST antibodies shows binding of GST fused
SH3 domains with immobilized 29 aa N-terminal
extension of CoASy β or full-length CoASy α or
with control 6His-GST protein. The middle panel,
visualized with anti-CoASy antibodies, shows
immobilized on Ni-NTA full-length 6His-CoASy α
protein. The low panel, visualized with anti-6His
antibodies shows, immobilized on Ni-NTA
6His-GST-29 aa N-terminal extension of CoASy β
and 6His-GST protein
Summary of SH3 domains tested in the pull down experiment as direct interacting partners of CoASy β N-terminal extension and full
size CoAsy α protein
Tested SH3 domains
Binding to
N-terminal extension
of CoASy β
Binding to
CoASy α Protein function
Ruk1(SH3A) – –
Adapter protein involved in regulating diverse
signal transduction pathways
Ruk1(SH3B) – – –
Ruk1(SH3C) – – –
Grb2
– –
Adapter protein mediating coupling of activated growth
factor receptors with proline rich regions containing ligands
Spectrin + –
One of the key components of membrane skeleton
involved in membrane trafficking
c-Src – – Non-receptor tyrosine protein kinase of Src family
p67phox
C-terminal
+ + NADPH oxidase activator 1
Fyn + – Non-receptor tyrosine protein kinase of Src family
Crk – – Adapter protein recruiting cytoplasmic
proteins to the vicinity of tyrosine kinase
Fgr – – Non receptor tyrosine kinase of Src family
PLCγ + –
Phospholipase Cγ; Hydrolyzes phosphatidylinositol 4,5-biphosohate
(PIP2) in response to various extracellular stimuli to produce two
second messengers, diacylglycerol and inositol 1,4,5-triphosphate
GAP(Ras) – – GTPase activating protein of Ras
Csk + – Non-receptor tyrosine protein kinase. Negatively regulates
Fyn and other non receptor Src family tyrosine kinases
washed. As a control, non-specific binding of
SH3-GST domains to Ni-NTA agarose along with
immobilized GST-6His was tested for each SH3
domain. Interaction of SH3-GST domains with
immobilized proteins was analysed by Western blot
with GST specific antibodies.
Specific binding of different SH3 domains with
N-terminal 29 aa extension of CoASy β and/or
full-length CoAsy α was detected (Fig. 3). Table
summarizes results obtained in the pull down
experiment. We showed direct specific in vitro
interactions of N-terminal extension CoASy β with SH3
domains of Src family non-receptor tyrosine kinases
Fyn and Csk, phospholipase Cγ, NADPH oxidase
activator 1 – p67phox, and structural membrane
skeleton protein spectrin. Using GST-fused C-terminal
SH3 domain of p67phox we showed that CoASy α can
also directly interact with SH3 domains, and, contrary
to N-terminal 29 aa proline rich sequence, has several
separate proline rich motives. These data indicate that
p67phox protein may have several binding sites on
CoASy molecule, one of them located on N-terminus of
CoASy β and others – within the shared part of CoASy
α and β.
Noteworthy, in this experiment we confirmed
interaction predicted by Scansite program for PLCγ
SH3 domain. However, we failed to detect interactions
between CoASy and SH3 domains of tyrosine kinases
Src, Crk, and adapter protein Grb2 predicted by
ScanSite.
Although this type in vitro interaction experiments
has to be validated for in vivo systems in
co-immunoprecipitation experiments, we consider this
approach to be suitable for primary screening of protein
interactions. Our data indicate definitely that CoA
Synthase isoforms can potentially form complexes with
SH3 containing proteins involved in signal
transduction in cell. Furthermore, CoASy β isoform has
an additional N-terminal proline rich peptide. As it was
demonstrated here, this proline rich peptide
substantially extends the range of SH3 domains which
can bind directly to CoASy. Whether these interactions
interfere with regulation of the rate of Coenzyme A
biosynthesis, intracellular localization of CoASy or
with other intracellular functions remains to be
elucidated.
Acknowledgments. Oksana Breus was supported by
FEBS Collaborative Experimental Scholarships for
Central & Eastern Europe.
О. С. Бре ус, Г. Г. Па на сюк, І. Т. Гут, В. В. Філо нен ко,
І. О. Не ма за ний
Зба га чені проліном об ласті ко ен зим А-син таз α та β
взаємодіють з SH3 до ме на ми сиг наль них білків in vitro
Ре зю ме
Ко фер мент А-син та зи α та β містять зба га чені проліном
ділян ки, які мо жуть спри я ти їхньому ком плек со ут во рен ню з
білка ми, що містять SH3 до ме ни. Для пе ревірки цієї гіпо тези
ми про ве ли ек спе ри мен ти in vitro по зв’я зу ван ню по вно -
розмірно го білка КоА-син тази α та спе цифічно го для КоА-син -
та зи β N-кінце во го пеп ти ду із SH3 до ме на ми сиг наль них білків.
Ви я ви лося, що спе цифічний для КоА-син та зи β N-кінце вий пеп -
тид, ба га тий на пролін, спе цифічно взаємодіє з SH3 до ме на ми
ти ро зи но вих про теїнкіназ Fyn і CSK, фос фоліпази C γ, ак ти ва -
то ра NADPH окси да зи 1 – p67phox і ци тос ке лет но го білка
спек три ну. Крім того, SH3 до мен p67phox зв’я зується із зба га -
че ною проліном послідовністю, спільною для обох ізо форм.
Отри мані ре зуль та ти доз во ля ють при пус ти ти, що КоА-cин -
та зи пе ре бу ва ють у ком плек сах з сиг наль ни ми білка ми і, та -
ким чи ном, відбу вається ре гу ляція їхньої фер мен та тив ної
ак тив ності або ж КоА-син та зи невідо мим на да ний час спо со -
бом мо ду лю ють певні ета пи пе ре да ван ня сиг налів у клітині.
Клю чові сло ва: КоА син та за, SH3 до мен, об ласті зба га чені
проліном, сиг нальні білки.
О. С. Бре ус, Г. Г. Па на сюк, И. Т. Гут, В. В. Фи ло нен ко,
И. А. Не ма за ный
Обо га щен ные про ли ном об лас ти ко фер мент А-син таз α и β
вза и мо де йству ют с SH3 до ме на ми сиг наль ных бел ков
в усло ви ях in vitro
Ре зю ме
Ко фер мент А-син та зы α и β со дер жат бо га тые про ли ном об -
лас ти, воз мож но, спо со бству ю щие их ком плек со об ра зо ва нию
с бел ка ми, со дер жа щи ми SH3 до ме ны. Для про вер ки этой ги по -
те зы мы про ве ли экс пе ри мен ты по свя зы ва нию in vitro по лно -
раз мер но го бел ка КоА-син та зы α и спе ци фи чес ко го для
КоА-син та зы β N-кон це во го пеп ти да с SH3 до ме на ми сиг наль -
ных бел ков. Ока за лось, что про лин-бо га тый N-кон це вой пеп -
тид КоА-син та зы β спе ци фи чес ки вза и мо де йству ет с SH3
до ме на ми ти ро зи но вых про те ин ки наз Fyn и CSK, фос фо ли па -
зы Cγ, ак ти ва то ра NADPH окси да зы 1 – p67phox и бел ка ци -
тос ке ле та спек три на. Кро ме это го, SH3 до мен p67phox
вза и мо де йству ет с об ога щенной про ли ном по сле до ва тель нос -
тью, об щей для об е их изо форм. По лу чен ные ре зуль та ты по -
зво ля ют пред по ло жить, что КоА-син та зы α и β об ра зу ют
ком плек сы с сиг наль ны ми бел ка ми и, та ким об ра зом, ре гу ли -
руется фер мен та тивная ак тив ность КоА-син таз либо же
КоА-син та зы не из вес тным на дан ный мо мент спо со бом моду -
ли ру ют опре де лен ные эта пы пе ре да чи сиг на лов в клет ке.
Клю че вые сло ва: КоА-cин та за, SH3 до мен, об лас ти, об ога -
щен ные про ли ном, сиг наль ные бел ки.
127
PROLINE RICH REGIONS COENZYME A SYNTHASE α AND β INTERACT WITH SH3
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УДК 577.2, 577.1
Надійшла до ре дакції 30.10.07
128
BREUS O. S. ET AL.
|
| id | nasplib_isofts_kiev_ua-123456789-157672 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-11-24T04:44:46Z |
| publishDate | 2008 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Breus, O.S. Panasyuk, G.G. Gout, I.T. Filonenko, V.V. Nemazanyy, I.O. 2019-06-20T16:52:13Z 2019-06-20T16:52:13Z 2008 Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro / O.S. Breus, G.G. Panasyuk, I.T. Gout, V.V. Filonenko, I.O. Nemazanyy // Біополімери і клітина. — 2008. — Т. 24, № 2. — С. 123-128. — Бібліогр.: 16 назв. — англ. 0233-7657 http://dx.doi.org/10.7124/bc.000799 https://nasplib.isofts.kiev.ua/handle/123456789/157672 577.2, 577.1 Coenzyme A-synthases α and β (CoASy α and CoASy β ) contain proline rich regions which may bring them into complexes with SH3-domain containing proteins. To test whether CoASy isoforms can bind to SH3 domains we performed in vitro pull down experiments. It was found that CoASy β N-terminal extension, which is especially abundant in prolines, can interact specifically and directly with SH3 domains of tyrosine kinases Fyn and CSK, phospholipase Cγ, NADPH oxidase activator 1 – p67phox, and cytoskeleton protein spectrin. Furthermore, C-terminal SH3 domain of p67phox can also interact with SH3 binding site that resides on the shared part of CoASyβ and CoASyα . These data demonstrated that CoA Synthases could be involved in complexes with signaling proteins in living cells which may regulate enzymatic activities of CoA Synthases or vice versa CoA Synthase may modulate some steps in signal transduction in the cell in currently unknown way. Кофермент А-синтази α та β містять збагачені проліном ділянки, які можуть сприяти їхньому комплексоутворенню з білками, що містять SH3 домени. Для перевірки цієї гіпотези ми провели експерименти in vitro по зв’язуванню повнорозмірного білка КоА-синтази α та специфічного для КоА-синтази β N-кінцевого пептиду із SH3 доменами сигнальних білків. Виявилося, що специфічний для КоА-синтази β N-кінцевий пептид, багатий на пролін, специфічно взаємодіє з SH3 доменами тирозинових протеїнкіназ Fyn і CSK, фосфоліпази Cγ , активатора NADPH оксидази 1 – p67phox і цитоскелетного білка спектрину. Крім того, SH3 домен p67phox зв’язується із збагаченою проліном послідовністю, спільною для обох ізоформ. Отримані результати дозволяють припустити, що КоА-cинтази α і β перебувають у комплексах з сигнальними білками і, таким чином, відбувається регуляція їхньої ферментативної активності або ж КоА-синтази невідомим на даний час способом модулюють певні етапи передавання сигналів у клітині. Кофермент А-синтазы α и β содержат богатые пролином области, возможно, способствующие их комплексообразованию с белками, содержащими SH3 домены. Для проверки этой гипотезы мы провели эксперименты по связыванию in vitro полноразмерного белка КоА-синтазы α и специфического для КоА-синтазы β N-концевого пептида с SH3 доменами сигнальных белков. Оказалось, что пролин-богатый N-концевой пептид КоА-синтазы βспецифически взаимодействует с SH3 доменами тирозиновых протеинкиназ Fyn и CSK, фосфолипазы Cγ, активатора NADPH оксидазы 1 – p67phox и белка цитоскелета спектрина. Кроме этого, SH3 домен p67phox взаимодействует с обогащенной пролином последовательностью, общей для обеих изоформ. Полученные результаты позволяют предположить, что КоА-синтазы α и β образуют комплексы с сигнальными белками и, таким образом, регулируется ферментативная активность КоА-синтаз либо же КоА-синтазы неизвестным на данный момент способом модулируют определенные этапы передачи сигналов в клетке. en Інститут молекулярної біології і генетики НАН України Біополімери і клітина Структура та функції біополімерів Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro Обогащенные пролином области кофермент А-синтаз α и β взаимодействуют с SH3 доменами сигнальных белков в условиях in vitro Збагачені проліном області коензим А-синтаз α та β взаємодіють з SH3 доменами сигнальних білків in vitro Article published earlier |
| spellingShingle | Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro Breus, O.S. Panasyuk, G.G. Gout, I.T. Filonenko, V.V. Nemazanyy, I.O. Структура та функції біополімерів |
| title | Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro |
| title_alt | Обогащенные пролином области кофермент А-синтаз α и β взаимодействуют с SH3 доменами сигнальных белков в условиях in vitro Збагачені проліном області коензим А-синтаз α та β взаємодіють з SH3 доменами сигнальних білків in vitro |
| title_full | Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro |
| title_fullStr | Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro |
| title_full_unstemmed | Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro |
| title_short | Proline rich regions of coenzyme A synthase α and β interact with SH3 domains of signaling proteins in vitro |
| title_sort | proline rich regions of coenzyme a synthase α and β interact with sh3 domains of signaling proteins in vitro |
| topic | Структура та функції біополімерів |
| topic_facet | Структура та функції біополімерів |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/157672 |
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