Generation of GFP-SMAD cell lines.
A, SMAD protein phylogeny. Clustering analysis and systemic
representation of SMAD proteins with the indicated domains (MH1, MH2,
and SAD). SMAD categorization is indicated on the right.
B,
SMAD cell line time series after doxycycline induction was analyzed on
immunoblot. HeLa cell lines were induced with 1 μg/ml of doxycycline for
4, 8, 16, 20, or 24 h prior to harvesting or harvested without
induction. Blots were probed using GFP or GAPDH antibodies.
Discussion
SMAD
signaling is involved in many different cellular processes. In this
study interactome datasets for all SMAD proteins were generated using
affinity purification for HeLa cells followed by mass spectrometry
analysis. The nuclear transport protein IPO5 was identified as a novel
interactor for BMP-regulated SMAD1 and using confocal microscopy a
functional link was shown between IPO5 and the BMP-regulated R-SMADs but
not TGF-β-regulated R-SMADs. By extending the NLS lysine stretch in
SMAD3 we found SMAD3 nuclear localization sensitive to IPO5, suggesting
that the length of the lysine stretch in the NLS is responsible for
differentiation between the BMP- and TGF-β-regulated SMADs.
SMAD proteins shuttle continuously between the cytoplasm and nucleus, which is independent of a receptor activation signal (
9).
XPO1 (2p15), CRM1, Exportin 1.
Phosphorylation of R-SMAD followed by SMAD4
recruitment causes nuclear accumulation of SMADs, which result in
reduced nuclear export.
Transcription factors like TAZ, Tafazzin (Xq28) (
25)
have been shown to be
responsible for SMAD nuclear accumulation,
although the exact mechanism is unknown.
Conserv. domain: Lysophospholipid Acyltransferases (LPLATs) of Glycerophospholipid Biosynthesis: AGPAT-like
Lysophospholipid
acyltransferase (LPLAT) superfamily member: acyltransferases of de novo
and remodeling pathways of glycerophospholipid biosynthesis which
catalyze the incorporation of an acyl group from either acylCoAs or
acyl-acyl carrier proteins (acylACPs) into acceptors such as glycerol
3-phosphate, dihydroxyacetone phosphate or lyso-phosphatidic acid.
Included in this subgroup are such LPLATs as
1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT, PlsC), Tafazzin
(product of Barth syndrome gene), and similar proteins.
TITLE Defective Mitochondrial Cardiolipin Remodeling Dampens HIF-1alpha
Expression in Hypoxia
JOURNAL Cell Rep 25 (3), 561-570 (2018)
PUBMED 30332638
REMARK GeneRIF: Tafazzin deficiency is associated with defective
remodeling of the mitochondrial phospholipid cardiolipin causing
cardiomyopathy in Barth syndrome.
In the nucleus, phosphorylation
of R-SMAD complexes is gradually reduced causing dissociation of SMAD4
followed by export from the nucleus. This constant SMAD
nuclear/cytoplasmic cycling is an important control mechanism for
constant monitoring of the receptor activation status. This results in a
quick adaptation of nuclear SMAD accumulation, when the signal is
present and to reduce to steady state levels when the receptors become
inactive.
Initially,
transport proteins importin α1* (KBNA1) and
β1* (KBNB1) (
26)
have been connected with SMAD signaling.
Later studies implied IPO7** and
IPO8** in signal-dependent and -independent transport of SMAD1/3/4
involving NLS found in the MH1 domain.
Our results corroborate an
important role of IPO7 in subcellular distribution of SMAD1, -2, -3, -5,
and -9 ().
IPO7 seems to be the major contributor of SMAD nuclear import, whereas
the import mediated by IPO5 is restricted. Nuclear import of the R-SMADs
by IPO7 does not select for a specific type of R-SMAD ().
Systemically testing IPO5 against all SMADs in this study shows
selectivity of IPO5 for BMP-activated R-SMADs, which is determined by
the length of the lysine stretch of the NLS. Selectivity of the IPO5
protein for the cargo allows for regulation of signaling, thus adding a
new player in controlling the subcellular distribution of SMAD proteins.
TÄSSÄ sivuhyppy: Haen eri IPO proteiineja ja importiini alfa 1 ja importiini alfa 2 ja importiini beta proteiineja esiin. Merkkaan tähdeää ne joita on tarkoitettu tekstissä:
// IPO1*, IPOB, (17q21.32) Importin subunit B1*, Karyopherin subunit beta 1.
-
Functions in nuclear protein import, either in
association with an adapter protein, like an importin-alpha subunit,
which binds to nuclear localization signals (NLS) in cargo substrates,
or by acting as autonomous nuclear transport receptor. Acting
autonomously, serves itself as NLS receptor. Docking of the
importin/substrate complex to the nuclear pore complex (NPC) is mediated
by KPNB1 through binding to nucleoporin FxFG repeats and the complex is
subsequently translocated through the pore by an energy requiring,
Ran-dependent mechanism. At the nucleoplasmic side of the NPC, Ran binds
to importin-beta and the three components separate and importin-alpha
and -beta are re-exported from the nucleus to the cytoplasm where GTP
hydrolysis releases Ran from importin. The directionality of nuclear
import is thought to be conferred by an asymmetric distribution of the
GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus.
Mediates autonomously the nuclear import of ribosomal proteins RPL23A,
RPS7 and RPL5. Binds to a beta-like import receptor binding (BIB) domain
of RPL23A. In association with IPO7 mediates the nuclear import of H1
histone. In vitro, mediates nuclear import of H2A, H2B, H3 and H4
histones. In case of HIV-1 infection, binds and mediates the nuclear
import of HIV-1 Rev. Imports SNAI1 and PRKCI into the nucleus.
IPO2, TNPO1, Transportin 1, KPNB2 (5q13.2) MIP1, MIP, TRN.
IPO3, TNPO2, (19p13.13). Transportin 2. Karyopherin beta 2b, (KPNB2b)
IPO9
IPOA1,Pendulin,Karyopherin alpha 2,(KPNA2),(17q24.2), SRP1alpha, RAG cohort protein 1.
IPOA2
IPOA3, Importin subunit alpha 3,( KPNA4), Qip1, (3q25.33), Importin subunit alpha 3.
IPOA4, ( KPNA3), Qip2, (13q14.2).Importin subunit alpha 4.
IPOA5: (3q21.1) ( KPNA1), Karyopherin subunit alpha 1, Importin subunit alpha 5. RAG cohort protein 2, recombination activating gene cohort 2.
The transport of molecules between the nucleus
and the cytoplasm in eukaryotic cells is mediated by the nuclear pore
complex (NPC), which consists of 60-100 proteins. Small molecules (up to
70 kD) can pass through the nuclear pore by nonselective diffusion
while larger molecules are transported by an active process. The protein
encoded by this gene belongs to the importin alpha family, and is
involved in nuclear protein import. This protein interacts with the
recombination activating gene 1 (RAG1) protein and is a putative
substrate of the RAG1 ubiquitin ligase. Alternative splicing results in
multiple transcript variants. [provided by RefSeq, Nov 2012]
( Näitä näyttää olevan runsaasti Gene Cards lähteessä ja monen yhteydssä mainitaan viruksista) -//
The SMAD activation pathway is a signaling cascade that gains complexity in regulation over different levels in the cell (
14,
27,
–32).
Over 30 different molecules including TGF-β and BMP can be found in the human body that belong to the TGF-β superfamily (33).
These molecules can signal to a combination of seven type I and five
type II receptors. Because not all of these receptors are present in
each cell type this makes TGF-β and BMP signaling very
context-dependent. It is important to note that we did not identify any
TGF-β or BMP receptor in GFP-SMAD purifications from the cytoplasmic
fractions, which could be due to the low efficiency of membrane protein
extraction in our procedures.
The N-terminal location of the GFP moiety in the SMAD fusion constructs was chosen to maintain the C-terminal SSXS residues required for R-SMAD phosphorylation and protein activation (6,–8).
This resulted in fusion constructs that could be phosphorylated upon
stimulation of their receptors using their respective ligands ().
However, the possibility still remains that interaction partners have
been missed due to N-terminal tagging of SMAD proteins.
In our
experiments we confirmed binding of the transcriptional repressor LEMD3
to SMAD3 in the nucleus and provide evidence that LEMD3 and SMAD4
binding are mutually exclusive ().
LEMD3 (12q14.3), Inner nuclear membrane protein Man1.tekee interaktion SMAD1,2,3 ja 5 kanssa ja sitoutuu sekä fosforyloituun että fosforyloitumattomaan R-SMADiin.
Interestingly, no LEMD3 binding to other R-SMADs is observed, whereas
SKI and SKIL are identified in SMAD2/3 and -4, which indicates a high
specificity in repressor interactions.
Activated
SMAD proteins stimulate gene transcription, but the exact nature of
their nuclear coactivators has not been determined in much detail. We
provide evidence that Mediator, TFIID, and SET/MLL but not BAF complex
could participate in transcription activation by the SMAD1 and SMAD2
proteins. The observation of specific co-activators for specific SMAD
family members warrants a further study into the downstream
transcription activation pathways for TGF-β and BMP signaling.
( Alla oleva artikkeli selvittää
SMAD3:n osuuta mikroRNA-säätelyssä, esimerkki on munuaisfibroosin terapiasta:
Article in Frontiers in Physiology · March 2015
DOI: 10.3389/fphys.2015.00082 ·)
