Liu, J., Prickett, T.D., Elliott, E., Meroni, G. & Brautigan, D.L. Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit 4. Proc. Natl Acad. Sci. USA 98, 6650-6655

ArticleinProceedings of the National Academy of Sciences 98(12):6650-5 · July 2001with24 Reads
Impact Factor: 9.67 · DOI: 10.1073/pnas.111154698 · Source: PubMed
Abstract

Opitz syndrome (OS) is a human genetic disease characterized by deformities such as cleft palate that are attributable to defects in embryonic development at the midline. Gene mapping has identified OS mutations within a protein called Mid1. Wild-type Mid1 predominantly colocalizes with microtubules, in contrast to mutant versions of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid screening, we found that the alpha4-subunit of protein phosphatases 2A/4/6 binds Mid1. Epitope-tagged alpha4 coimmunoprecipitated endogenous or coexpressed Mid1 from COS7 cells, and this required only the conserved C-terminal region of alpha4. Localization of Mid1 and alpha4 was influenced by one another in transiently transfected cells. Mid1 could recruit alpha4 onto microtubules, and high levels of alpha4 could displace Mid1 into the cytosol. Metabolic (32)P labeling of cells showed that Mid1 is a phosphoprotein, and coexpression of full-length alpha4 decreased Mid1 phosphorylation, indicative of a functional interaction. Association of green fluorescent protein-Mid1 with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Mid1 association with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase that is targeted specifically to Mid1 by alpha4. Human birth defects may result from environmental or genetic disruption of this regulatory cycle.

Full-text

Available from: Todd D Prickett
Phosphorylation and microtubule association of the
Opitz syndrome protein mid-1 is regulated by
protein phosphatase 2A via binding to the
regulatory subunit
4
Jun Liu*, Todd D. Prickett*, Elizabeth Elliott*, Germana Meroni
, and David L. Brautigan*
*Center for Cell Signaling, University of Virginia School of Medicine, P.O. Box 800577, Charlottesville, VA 22908-0577; and
Telethon Institute of Genetics
and Medicine (TIGEM), Via P. Castellino 111, 80131 Naples, Italy
Communicated by Edmond H. Fischer, University of Washington, Seattle, WA, March 29, 2001 (received for review February 20, 2001)
Opitz syndrome (OS) is a human genetic disease characterized by
deformities such as cleft palate that are attributable to defects in
embryonic development at the midline. Gene mapping has identified
OS mutations within a protein called Mid1. Wild-type Mid1 predom-
inantly colocalizes with microtubules, in contrast to mutant versions
of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid
screening, we found that the
4-subunit of protein phosphatases
2A46 binds Mid1. Epitope-tagged
4 coimmunoprecipitated en-
dogenous or coexpressed Mid1 from COS7 cells, and this required
only the conserved C-terminal region of
4. Localization of Mid1 and
4 was influenced by one another in transiently transfected cells.
Mid1 could recruit
4 onto microtubules, and high levels of
4 could
displace Mid1 into the cytosol. Metabolic
32
P labeling of cells showed
that Mid1 is a phosphoprotein, and coexpression of full-length
4
decreased Mid1 phosphorylation, indicative of a functional interac-
tion. Association of green fluorescent protein–Mid1 with microtu-
bules in living cells was perturbed by inhibitors of MAP kinase
activation. The conclusion is that Mid1 association with microtubules,
which seems important for normal midline development, is regulated
by dynamic phosphorylation involving MAP kinase and protein phos-
phatase that is targeted specifically to Mid1 by
4. Human birth
defects may result from environmental or genetic disruption of this
regulatory cycle.
T
he Opitz GBBB syndrome (OS), also known as the hypospa-
dias–dysphagia syndrome or telecanthus with associated ab-
normalities, is associated with midline abnormalities such as cleft
lip, laryngeal cleft, heart defects, hypertelorism, hypospadias, im-
perforate anus, agenesis of the corpus callosum, and developmental
delay (1). It was demonstrated that OS is a heterogeneous disorder,
with X linked and autosomal forms. No phenotypic differences
between the two linkage types were discerned except that ante-
verted nares and posterior pharyngeal cleft were seen only in the X
linked form (2).
The study (1) of a family in which OS segregated with an X
chromosome inversion revealed the position of the gene on Xp22,
which was referred to as Mid1 (for midline-1). The Mid1 gene
encodes a 667-aa protein that is expressed ubiquitously in both
embryonic and adult tissues. The prominent expression of Mid1 in
undifferentiated cells in the central nervous, gastrointestinal, and
urogenital systems suggested that abnormal cell proliferation
andor migration may underlie the defect in midline development
characteristic of OS (3).
The Mid1 protein belongs to a RING finger family of nuclear
factors that contain protein–protein interaction domains and have
been implicated in fundamental processes such as body-axis pat-
terning and cell transformation. Besides the N-terminal RING
finger domain, Mid1 also contains four additional domains: two
potential zinc-binding B box domains, a leucine coiled-coil domain
characteristic of the ‘‘RING-B box-coiled coil’’ subgroup of RING
finger proteins, and a RFP-like C-terminal domain. OS-associated
mutations have been identified in both C- and N-terminal regions
of Mid1 (2).
Previous studies showed that Mid1 is a microtubule-associated
protein that may influence microtubule dynamics in Mid1-
overexpressing cells (4, 5). Mid1 is associated with microtubules
throughout the cell cycle, colocalizing with cytoplasmic fibers in
interphase and with mitotic spindle and midbodies during mitosis
and cytokinesis (5). Overexpressed Mid1 proteins harboring C-
terminal mutations described in OS patients lack the ability to
associate with microtubules, forming cytoplasmic clumps instead
(4). Thus, the inability to associate with microtubules is one possible
molecular mechanism underlying the OS phenotype. However, the
cellular function of Mid1 and the mechanism to control Mid1
subcellular localization are still unknown.
The
4 protein originally was discovered as a phosphoprotein
associated with Ig receptor in B cells (6). It shares 28% sequence
identity with the yeast Tap42 protein, which functions as part of the
yeast target of rapamycin (TOR)-signaling pathway (7–12). Yeast
Tap42 binds protein phosphatases Sit4 and Pph2122 that corre-
spond to mammalian PP6 and PP2A, respectively (7), and binding
is independent of Tpd3, the PP2A yeast A subunit (7, 12). Likewise,
mammalian
4 associates with the C subunit of PP2A in the absence
of A (PR65) subunit (13, 14). In yeast, Tap42-phosphatase associ-
ation has been shown to be rapamycin-sensitive (7), consistent with
a report of TOR-mediated phosphorylation of Tap42 (12). Al-
though this pathway presumably links nutrient status to protein
synthesis, the targets of Tap42 phosphatases have not been eluci-
dated. Overexpression of mouse FLAG-
4 in COS cells promoted
4-phosphatase formation and caused dephosphorylation of elon-
gation factor-2, which is activated by dephosphorylation (15). In the
present study, we set out to identify proteins that bind directly to
4
and, therefore, might be cellular substrates of this form of phos-
phatase. Yeast two-hybrid screening with mouse
4 as bait yielded
multiple clones of Mid1 as a strong interactor. We demonstrate that
the C-terminal 120 residues of
4 are sufficient for binding to Mid1
and expression of FLAG-
4 in cells promotes the dephosphoryla-
tion of Mid1 and dissociation of Mid1 from microtubules. This
uncovers a novel function for
4 phosphatase in the regulation of
Mid1 localization and posits a role for
4 in midline development.
Materials and Methods
Two-Hybrid Screen. The screen was done by using full-length mu-
rine-
4 in pGBT10 vector (BamHI-EcoRI), a derivative of pGBT9
designed and provided by Ian Macara (University of Virginia), that
Abbreviations: OS, Opitz syndrome; Mid1, midline-1; GFP, green fluorescent protein; TOR,
yeast target of rapamycin.
To whom reprint requests should be addressed. E-mail: db8g@virginia.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
66506655
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June 5, 2001
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no. 12 www.pnas.orgcgidoi10.1073pnas.111154698
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contains the Gal4 DNA-binding domain. Bait was transformed into
the yeast strain HF7c by using the lithium acetate method and
grown further in synthetic medium lacking tryptophan (16). The
library screen was done by using a 9-day embryonic mouse cDNA
library cloned into pVP16 (created by Stan Hollenberg, Fred
Hutchinson Cancer Center, Seattle). Library transformation was
done in a sequential manner, such that 6 10
6
independent clones
were tested and plated on synthetic medium containing 10 mM
3-aminotriazole (Sigma) and lacking histidine, leucine, and trypto-
phan. Positive clones were determined by growth on plates and
-galactosidase filter assays. Clones were rescued by electropora-
tion into Escherichia coli HB101 and grown on M9 plates lacking
leucine, which allowed for analysis of positives by transformation
tests and DNA sequencing.
Plasmids and Mutagenesis. Mid1 full-length cDNA was derived from
c-Mycgreen fluorescent protein (GFP)-Mid1 (5) by PCR ampli-
fication. GFP-Mid1 was constructed by placing Mid1 cDNA in-
frame in the EcoRI site of pEGFP-C3 vector. FLAG-
4 was
constructed as described previously (13). FLAG-
4(1–111),
FLAG-
4(1–249), and all mutated forms of GFP-Mid1 were
generated by using the Stratagene Quick Change Mutagenesis Kit
according to the manufacturer’s protocol. FLAG-
4(220–340) was
obtained by PCR as described previously (17).
Cell Culture, Transfection, Immunoprecipitation, and Immunoblotting.
COS7 cells were maintained in DMEM (Life Technologies, Gaith-
ersburg, MD) containing 10% newborn calf serum (Life Technol-
ogies) at 37°C, 5% CO
2
. Cells were grown to 5060% confluency
and transfected for 18 h by using FuGene 6 reagent (Roche
Diagnostics) according to the manufacturer’s instructions. Usually,
cells in 60-mm dishes were transfected with 2
g of plasmid DNA,
and 1
g of DNA was used for cells in 35-mm dishes. Immuno-
precipitation with anti-FLAG M2 affinity gel (Sigma) or anti-GFP-
conjugated Agarose (Santa Cruz ) and immunoblotting were done
as described previously (18).
Metabolic [
32
P] Labeling. COS7 cells in 60-mm dishes were trans-
fected for 18 h and then incubated for3hinphosphate-free DMEM
supplemented with dialyzed newborn calf serum. Cells then were
labeled for 1.5 h in the same medium containing 1 mCiml
[
32
P]orthophosphate (NEN).
Fluorescence Microscopy. COS7 cells were grown on fibronectin-
coated coverslips in 35-mm dishes. After transfection, cells were
washed with 1.2 PEM buffer (120 mM Pipes, pH 7.06mM
EGTA2.4 mM MgCl
2
) and then fixed with 3.5% paraformalde-
hyde for 15 min. FLAG-
4 and microtubules were stained with
anti-FLAG M2 and anti-
-tubulin antibodies plus a tetramethyl-
rhodamine B isothiocyanate-labeled secondary antibody (Sigma).
Nuclei were stained by using the DNA-specific stain 4,6-diamidino-
2-phenylindole (DAPI). Images were acquired on a Nikon Micro-
phot-SA equipped with a Hamamatsu C4742 charge-coupled de-
vice camera driven by
OPENLAB 2.0.6 (Improvision, Lexington, MA)
software. Images were processed in Adobe
PHOTOSHOP 5.5 (Adobe
Systems, Mountain View, CA).
GFP-Mid1 Localization in Living Cells
.
COS7 cells were plated onto
fibronectin-coated 22 22-mm coverglasses and transiently trans-
fected with GFP-Mid1. Cultures were incubated overnight at 37°C.
Each coverglass was placed into a metal chamber filled with 1 ml
of serum-free, phenol red-free DMEM on a warmed stage. Images
were captured before and at 10 and 30 min after treatment with
either 10
M U0126 (LC Laboratories, Woburn, MA) or 10
M
PD98059 (Upstate Biotechnology, Lake Placid, NY) by using a
Zeiss Axiovert 135TV inverted microscope equipped with a GFP
filter set (excitation 475 nm, emission 505 nm) from Omega
Optical (Brattleboro, VT) and a Hamamatsu (Middlesex, NJ)
C4742 charge-coupled device camera driven by
INNOVISION
(Durham, NC) software. Images were processed in Adobe PHOTO-
SHOP 5.5 (Adobe Systems).
Results and Discussion
We screened a yeast two-hybrid library derived from mouse embryo
by using as bait murine full-length
4, the putative mammalian
homologue of yeast Tap 42. From 6 million clones we isolated 200
positives by growth on selective medium, and 65 of these showed
expression of
-galactosidase as a reporter. All 65 clones were
sequenced, and, of these, 20 were the catalytic subunit of PP2A, 4
were PP4, and another 8 were identical to regions of the Mid1 gene
product (1). The Mid1 fragments identified in the
4 screen all
contained the region from Thr58 to Asp180, comprising the B box
domains, but lacked the N- and C-terminal flanking regions such as
Ring finger and coiled-coil domains. We found that Mid1(58–180)
alone had no transactivating activity. In the two-hybrid system,
pairwise transformation with
4 plus the C subunit of PP2A was
strongly positive and Mid1(58–180) gave comparable growth
(shown as a darkened area by densitometry in Fig. 1a). We also
show (Fig. 1a) that PP6, the mammalian homologue of yeast Sit4,
interacted with
4 in a two-hybrid assay, consistent with an earlier
report (19). By contrast, PP1c that is about 50% identical in
sequence to PP2A but does not bind
4 (13) was a negative control
to show specificity of interaction, and, indeed, no growth was
observed (Fig. 1a).
Association of Mid1,
4, and PP2A in Living Cells. We expressed
FLAG-tagged mouse Mid1(58–180) protein with or without myc-
tagged
4 in COS7 cells and immunoprecipitated with anti-FLAG
antibody (Fig. 1b). FLAG-Mid1(58–180) coprecipitated endoge-
nous
4 (lane 2). Endogenous
4 and, much more, myc-
4 were
a
b
c
Fig. 1. Association of
4 with Mid1 and PP2A. (a) Yeast two-hybrid analysis
showed that strains expressing Gal4-DBD-
4 grew on triple-drop out media (trp-,
leu-, his-) only when also expressing PP2A, PP6, or Mid1 (residues 58–180),
indicative of an interaction with
4. Controls included single transformants and
PP1C that did not support growth, because it does not bind
4. The plate was
scanned on a densitometer to show growth of yeast colonies. (b) COS7 cells were
transfected with FLAG-tagged Mid1(58–180) with or without myc-tagged
4.
Cells were lysed and immunoprecipitation was performed by using anti-FLAG M2
affinity gel. FLAG-Mid1(58 –180) and
4 were detected in the immunoprecipi-
tates by immunoblotting with anti-FLAG and anti-
4 antibodies. When present,
myc-
4 was detected by anti-myc immunoblotting. (c)COS7 cells transfected with
vector alone, FLAG-
4 (full-length), FLAG-
4(1–249), FLAG-
4(220–340), or
FLAG-
4(1–111) were lysed, and anti-FLAG immunoprecipitation was performed.
FLAG-
4 coprecipitated endogenous Mid1 and PP2A-C, detected by anti-FLAG,
anti-Mid1, and anti-PP2A-C immunoblotting.
Liu et al. PNAS
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coprecipitated with FLAG-Mid1(58–180) (lane 4). Conversely,
FLAG-
4 was expressed in COS7 cells, and it coprecipitated
specifically the endogenous, 80-kDa Mid1 and 36-kDa C subunit of
PP2A (Fig. 1c, second lane). The results showed that, in living cells,
FLAG-Mid1(58–180) bound
4 and that FLAG-
4couldbindto
both endogenous Mid1 and the C subunit of PP2A. Although from
these results one cannot tell whether all three proteins, Mid1,
4,
and PP2A, are bound together, other evidence (see below) suggests
such complexes can assemble in living cells.
We mapped the regions in
4 required for the binding to Mid1
and the C subunit of PP2A (Fig. 1c). Deletion mutants of FLAG-
4
were produced by PCR and expressed in COS7 cells. Analysis of
anti-FLAG immunoprecipitates showed that the C-terminal one-
third of
4, encompassing only amino acids 220–340, was sufficient
Fig. 2. Codependent distribution of GFP-Mid1 and FLAG-
4. (a) COS7 cells were fixed and stained with anti-
4 antibodies to show the distribution of the endogenous
4 protein in a nontransfected cell (first frame) and in a cell expressing GFP-Mid1 (second frame). The distribution of GFP-Mid1 itself is shown in green, and colocalization
of GFP-Mid1 and endogenous
4 is shown in yellow in the merged image (fourth frame). (b) COS7 cells were cotransfected to express both GFP-Mid1 and FLAG-
4.
Cells were fixed and GFP-Mid was visualized by direct fluorescence microscopy, and FLAG-
4 was stained as described. From multiple experiments, representative cells
with increasing levels of FLAG-
4 (columns 1, 2, and 3, left to right) were compared for localization of FLAG-
4 and GFP-Mid1. In the merged images, nuclei were stained
by DAPI. (c) Cells expressing both GFP-Mid1 and FLAG-
4 to give foci of GFP-Mid1 (green) were fixed and stained for
-tubulin (red) and DAPI (blue).
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for binding to Mid1 but failed to bind the C subunit of PP2A.
Conversely, the N-terminal two-thirds of
4, residues 1–249, bound
C subunit of PP2A but not Mid1. Further truncation of
4toa
region of residues 1–111 gave a protein that bound no PP2A C
subunit, indicating that a region between 111 and 249 of
4is
required for binding to PP2A. These results agree with the previous
assignment of the phosphatase-binding site in
4 (14). Our results
show that the C-terminal region of
4 binds Mid1. This is the region
of
4 most conserved between species. Distinct regions of
4bind
PP2A and Mid1, seemingly independent of one another.
Colocalization of
4 and GFP-Mid1 with Microtubules. Recent studies
have found that a Mid1-GFP fusion protein colocalized with
microtubules (4, 5). As expected, this GFP-Mid1 protein expressed
Fig. 3. OS mutants of GFP-Mid1 associate with FLAG-
4. (a) COS7 cells were trans-
fected to express GFP-Mid1(1–480) together with each of the following FLAG con-
structs: FLAG empty vector, FLAG-
4, and FLAG-
4(220–340). Cells were fixed and
GFP-Mid1 was visualized by direct fluorescence microscopy. FLAG-
4 was visualized by
anti-
4 immunofluorescence. Nuclei of the cells were visualized by DAPI staining. (b)
COS7 cells were cotransfected to express FLAG-
4 and different GFP-Mid1 fusion
constructs: GFP alone (as control), GFP-Mid1, GFP-Mid1(1–480), GFP-Mid1(L626P), and
GFP-Mid1(C266R). Immunoprecipitation was performed by using anti-FLAG M2 affin-
ity gel and FLAG-
4; GFP-Mid1 fusion proteins and PP2A were detected by immuno-
blotting with anti-FLAG, anti-GFP, and anti-PP2A. The mutant GFP-Mid1 proteins all
bound FLAG-
4.
Liu et al. PNAS
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in COS cells clearly and predominantly colocalized with the mi-
crotubule network (Fig. 2a, green). Endogenous Mid1 gave the
same microtubule localization, visualized by indirect immunofluo-
rescence (not shown). Endogenous
4 was stained as diffusely
cytoplasmic and in the nucleus by using anti-
4 antibodies (Fig. 2a,
first frame), and the same pattern was obtained with anti-FLAG
antibodies staining ectopically expressed FLAG-
4 (not shown).
However, in cells expressing GFP-Mid1, much of the endogenous
4 became colocalized with microtubules (Fig. 2a). Thus, expres-
sion of GFP-Mid1 caused recruitment of cytoplasmic
4 onto
microtubules. This also was observed when low levels of FLAG-
4
were coexpressed in COS cells with GFP-Mid1 (Fig. 2b, column 1).
On the other hand, the filamentous pattern of GFP-Mid1 on
microtubules was lost as more and more FLAG-
4 was produced
in cells by increasing the amount of DNA in transfections andor
the time of expression. Instead, GFP-Mid1 became distributed as
numerous, bright cytoplasmic foci (Fig. 2b, column 2) or a few large,
fluorescent clumps (see Fig. 2b, column 3). Over this range of
expression levels, FLAG-
4 alone was diffusely cytoplasmic (not
shown). As seen in the merged images, FLAG-
4 and GFP-Mid1
colocalized, even when their distribution changed. It is important to
note that even when essentially all the GFP-Mid1 was accumulated
into clumps, removal from microtubules by FLAG-
4 coexpression
did not have any apparent effect on the microtubules themselves,
based on immunofluorescent staining of
-tubulin in transfected
and nontransfected cells (Fig. 2c).
Redistribution of OS Mutant Forms of Mid1 by
4. Mutated Mid1
proteins found in OS patients show a tendency to form either
cytoplasmic foci or larger clumps (2). We expressed two OS mutant
Mid1 proteins, L626P and C226R, as GFP-Mid1 fusion proteins in
COS7 cells together with FLAG-
4. As seen in Fig. 3b, in anti-
FLAG immunoprecipitates both OS mutant GFP-Mid1 proteins
bound FLAG-
4 to the same extent as wild-type GFP-Mid1, so
these single-residue mutations did not interfere with the Mid1-
4
interaction. Therefore, the failure of these mutant Mid1 proteins to
colocalize with microtubules was not due simply to a loss of binding
to
4. Moreover, we produced GFP-Mid1(1–480), a deletion
mutant that lacks the entire B30.2 domain, to mimic the Mid1
C-terminal deletions seen in OS. This truncated GFP-Mid1 also
coimmunoprecipitated with FLAG-
4 (Fig. 3b). These data
showed that various OS mutations in Mid1 did not prevent binding
to
4. This conclusion is consistent with these regions not mapping
in the
4-binding segment of Mid1, obtained from the yeast
two-hybrid screen. Thus, Mid1 has separate sites for binding to
microtubules and to
4.
The B30.2 domain in the C-terminal region of Mid1 can be found
in many RING finger proteins as well as other unrelated proteins,
but its function is still poorly understood. The potential importance
of the B30.2 domain is implied by the concentration of mutations
in this region of Mid1 in OS patients. It has been proposed that the
RING-B box-coiled coil region is sufficient to mediate homodimer-
ization, and the C-terminal B30.2 domain has a key role in
microtubule association (5). Deletion of this domain from Mid1
eliminates microtubule association and results in the protein form-
ing clumps in the cytoplasm (2). We found that FLAG-
4 bound to
this truncated form of Mid1 (Fig. 3b, center lane). We transfected
COS7 cells to express GFP-Mid1(1–480) and, as expected, ob-
served accumulation of the GFP fusion protein in the perinuclear
region (Fig. 3a, left column). Coexpression of FLAG-tagged full-
length
4 with the GFP-Mid1(1–480) gave multiple clumps of
protein still in the perinuclear region, and, again (as in Fig. 2b), the
FLAG-
4 colocalized with the GFP-Mid1(1–480) (Fig. 3a, center
column). However, when FLAG-
4(220–340) was coexpressed, the
GFP-Mid1(1–480) was redistributed diffusely throughout the cy-
toplasm (Fig. 3a, right column). The nucleus appears pink in the
merged image because there was staining for FLAG-
4(220–340)
but not for GFP-Mid1(1–480) in the nucleus. Because FLAG-
4(220–340) binds Mid1 but not the phosphatase catalytic subunits
(see Fig. 1b), we expect that FLAG-
4(220–340) competed against
endogenous
4 for binding to GFP-Mid1(1–480) and thereby
displaced
4-associated phosphatase activity. The results suggest
that localization of Mid1 deleted in B30.2 domain is susceptible to
regulation by
4-phosphatase, probably involving phosphorylation
and dephosphorylation.
Phosphorylation–Dephosphorylation of Mid1. Could Mid1 be a phos-
phoprotein, and, if so, does binding to the
4-phosphatase reduce
a
b
c
Fig. 4. Dephosphorylation of GFP-Mid1 induced by
4 and MAP kinase activity
required for Mid1 association with microtubules. (a) COS7 cells were cotrans-
fected to express GFP-Mid1 together with either FLAG-
4 or FLAG empty vector.
Immunoprecipitation was performed by using anti-FLAG M2 affinity gel, and
FLAG-
4 and GFP-Mid1 were detected by anti-FLAG and anti-GFP antibodies. (b)
COS7 cells were transfected as described and radiolabeled with [
32
P]orthophos-
phate for 90 min. GFP-Mid1 was immunoprecipitated with anti-GFP antibodies
and was subjected to PhosphorImager analysis after SDSPAGE. Yield of GFP-
Mid1 in the immunoprecipitates was determined by anti-GFP immunoblotting.
FLAG-
4 in the cell lysate was detected by anti-FLAG immunoblotting. (c) COS7
cells were transfected to express GFP-Mid1, an initial image was captured (GFP-
:Mid1), and then the cells were treated with 10
M UO126 and images were
captured at the times indicated.
Fig. 5. Model for regulation of Mid1 association with microtubules by phos-
phorylation. The Mid1 protein is shown as two domains: a larger, elongated
N-terminal domain and a smaller, circular C-terminal domain that is either trun-
cated or contains mutations in OS (cross-hatched). Mid1 binds to other proteins
into high M
r
complexes in the cytoplasm (large circle) or can bind to microtubules
(rods shown on left side). The binding to microtubules is affected by MAP kinase,
which is inhibited by compounds UO126 and PD98059, and by binding to
4
(small, yellow circle) that associates with protein phosphatase catalytic subunits
(labeled PP) that promote dephosphorylation of Mid1 and its release from
microtubules.
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Page 5
its phosphorylation? We transfected COS7 cells to express GFP-
Mid1 plus either empty FLAG vector or FLAG-
4. Anti-FLAG
antibodies specifically coimmunoprecipitated FLAG-
4 with GFP-
Mid1 and PP2A, detected by immunoblotting with anti-GFP (Fig.
4a) and anti-PP2A (not shown). These results are comparable to
Fig. 3b but also show that coexpression of FLAG-
4 did not affect
the expression level of GFP-Mid1. Separately, cells were trans-
fected the same, labeled with
32
P
i
, and GFP-Mid1 protein was
recovered by anti-GFP immunoprecipitation. PhosphorImager
analysis showed that GFP-Mid1 was a
32
P-phosphoprotein (Fig. 4b).
Coexpression of FLAG-
4 decreased the
32
P labeling of GFP-Mid1
by almost half, in two independent experiments, without noticeable
effect on the GFP-Mid1 expression level. Our interpretation of
these results is that
4 targets phosphatase activity to Mid1, thereby
promoting Mid1 dephosphorylation in living cells.
Only one site of phosphorylation is predicted in Mid1, based on
searching for sequence motifs recognized by known kinases (http:兾兾
expasy.cbr.nrc.catools). Ser96 in a P-N-S-P sequence is a possible
substrate for MAP kinase (http:兾兾www.gcg.com). Xenopus nu-
clear factor-7 (XNF-7) is a B box protein with sequence similarity
to Mid1 (20). XNF-7 functions in Xenopus dorsal–ventral pattern-
ing (21), and its nuclear vs. cytoplasmic localization during oocyte
maturation is regulated by MAP kinase phosphorylation (20). The
phosphorylation site in XNF-7 for MAP kinase aligns with Ser96
in Mid1, so we predicted Mid1 would be a substrate for MAP
kinase. To test this hypothesis, we treated cells expressing GFP-
Mid1 with UO126 or PD98059 to inhibit MAP kinase activation.
Microscopic recording of live cells showed partial redistribution of
the GFP-Mid1 from microtubules to a punctate cytosolic localiza-
tion within 30 min of treatment with UO126 (Fig. 4c). Similar
results were obtained with cells treated with PD98059 (not shown).
The results show that MAP kinase activity was required to maintain
Mid1 association with microtubules.
These data support a model (Fig. 5), with phosphorylation of
Mid1 regulating its interaction with microtubules and, possibly, with
other proteins. The C-terminal B30.2 domain is required for
microtubule association, and increased phosphorylation of Mid1 is
proposed to enhance the affinity of Mid1 for microtubules or
microtubule-associated proteins. Conversely, dephosphorylation of
Mid1 by
4-associated phosphatases, probably PP2A, would reduce
the association of Mid1 with the microtubule network and result in
cytoplasmic localization. Phosphorylation may influence the bind-
ing of other partners to Mid1, because even forms of Mid1 deleted
in the B30.2 domain and, therefore, not bound to microtubules
showed redistribution when coexpressed with wild-type or ‘‘phos-
phatase-deficient’’ forms of
4.
Connecting Mid1 to Cellular Signals and Functions. The cellular
function of Mid1 is still unclear. Patients with mutations in Mid1
present with a variable array of malformations that are indicative of
a disturbance of primary midline development. Based on this, a
possible role for Mid1 has been postulated in the regulation of cell
proliferation, programmed cell death, cell migration, or even epi-
thelial–mesenchymal transformation. Our results offer evidence
that Mid1 is regulated by a phosphorylationdephosphorylation
mechanism. The potential ability of Mid1 to control cell behavior
might be triggered by a pathway that controls the function of
4 and
its associated phosphatases during development. Both
4 and its
yeast homologue, Tap42, associate with the C subunit of PP2A in
the absence of the PP2A PR65 or A subunit. In yeast, Tap42
association with phosphatases is sensitive to the macrocyclic-
immunosuppressive antibiotic rapamycin. This is consistent with
the report that TOR directly phosphorylates Tap42 and enhances
PP2A binding (12). In unpublished work, we have not seen phos-
phorylation of recombinant
4 by immunoprecipitated mTOR,
which otherwise is active in the assay with PHAS-1 as substrate. In
addition,
4 showed extremely low levels of
32
P labeling in meta-
bolically labeled cells, under a variety of conditions. Whether Tap42
and
4 both are controlled the same, e.g., by phosphorylation,
remains an open question. On the other hand, mutation of the
phosphorylation (Y307) and methylation (L309) sites in PP2A
greatly enhances binding to
4, so modifications of PP2A might
govern its association with
4 vs. the A subunit. Recently, yeast
TOR was shown to interact with Bik1, a microtubule-associated
protein required for microtubule assembly, stability, and function in
cell processes such as karyogamy and nuclear migration and
positioning (22, 23). Inhibition of TOR by rapamycin caused
significant defects in these cellular functions of yeast. Perhaps in
yeast, Tap42 and its bound phosphatase connect the TOR-signaling
pathway to the regulation of microtubule stability and function.
Previous studies showed that overexpression of Mid1 in mammalian
cells enhanced the stability of microtubules toward colcemid, a
microtubule-depolymerizing drug (4). A cycle of MAP kinase
phosphorylation and
4-PP2A dephosphorylation to regulate as-
sociation of Mid1 with microtubules opens new possibilities for
connecting signaling pathways to various functions of microtubules.
Learning more about the function of Mid1 and the role of its
phosphorylation hopefully will give insights into the developmental
defects seen in OS.
We thank Mary Foley for constant encouragement, members of the lab
for support and discussions, and Christine Palazzolo for assistance in
preparing the manuscript. We gratefully acknowledge grant support
from the U.S. Public Health Service–National Cancer Institute
(CA77584 to D.L.B.), the W. M. Keck Foundation (to D.L.B.), and the
March of Dimes Birth Defects Foundation (1-FY00-465 to G.M.).
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CELL BIOLOGY
Page 6
    • "Congenital abnormalities associated with human GLI3 are listed under the term " GLI3 morphopathies " , including Greig cephalopolysyndactyly syndrome (GCPS), non-syndromic polydactyly, Pallister Hall syndrome (PHS), acrocallosal syndrome, preaxial polydactyly type IV (PPD-IV) and postaxial polydactyly type A (PAPA) (Vortkamp et al. 1991; Kang et al. 1997; Radhakrishna et al. 1997 Radhakrishna et al. , 1999 Elson et al. 2002 ). Moreover, GLI3 is also associated with oral-facial-digital syndrome (OFDS) and Opitz syndrome (OS) (Liu et al. 2001; Johnston et al. 2010). A dominant developmental syndrome, GCPS with polydactyly and craniofacial abnormalities, is linked with large deletions, translocations and truncating mutations resulting in functional haploinsufficiency of GLI3 (Shin et al. 1999; Johnston et al. 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: The zinc-finger transcription factor GLI3 acts as a primary transducer of Sonic hedgehog (Shh) signaling in a context-dependent combinatorial fashion. GLI3 participates in the patterning and growth of many organs, including the central nervous system (CNS) and limbs. Previously, we reported a subset of human intronic cis-regulators controlling many known aspects of endogenous Gli3 expression in mouse and zebrafish. Here we demonstrate in a transgenic zebrafish assay the potential of two novel tetrapod-teleost conserved non-coding elements (CNEs) docking within GLI3 intronic intervals (intron 3 and 4) to induce reporter gene expression at known sites of endogenous Gli3 transcription in embryonic domains such as the central nervous system (CNS) and limbs. Interestingly, the cell culture based assays reveal harmony with the context dependent dual nature of intra-GLI3 conserved elements. Furthermore, a transgenic zebrafish assay of previously reported limb-specific GLI3 transcriptional enhancers (previously tested in mice and chicken limb buds) induced reporter gene expression in zebrafish blood precursor cells and notochord instead of fin. These results demonstrate that the appendage-specific activity of a subset of GLI3-associated enhancers might be a tetrapod innovation. Taken together with our recent data, these results suggest that during the course of vertebrate evolution Gli3 expression control acquired a complex cis-regulatory landscape for spatiotemporal patterning of CNS and limbs. Comparative data from fish and mice suggest that the functional aspects of a subset of these cis-regulators have diverged significantly between these two lineages.
    Full-text · Article · Oct 2015 · Development Growth and Regeneration
    1Comment 0Citations
    • "Alpha4 was shown to bind and induce a conformational change that inactivated the PP2Ac subunit, and this complex apparently protected the PP2Ac subunit from degradation [4], [21]. On the other hand, alpha4 is postulated to facilitate a MID1-alpha4-PP2Ac complex that promoted the ubiquitination of PP2Ac by MID1 [17], [24], [37]. Our ubiquitination data suggest that MID1 could interact with and target PP2Ac in the absence of alpha4 (Figure 2A). "
    [Show abstract] [Hide abstract] ABSTRACT: MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts.
    Full-text · Article · Sep 2014 · PLoS ONE
    0Comments 3Citations
    • "MID14PP2A complex is localized to microtubules via the interaction of MID1 with microtubule structures and is thought to be involved in the maintenance of microtubule stability (6, 20, 21). Although the precise function of this complex in microtubule stabilization remains unclear, it likely involves PP2A-mediated dephosphorylation of various MAPs. "
    [Show abstract] [Hide abstract] ABSTRACT: Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.
    Preview · Article · May 2012 · Journal of Biological Chemistry
    0Comments 15Citations
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