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
2A?4?6 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. Metabolic32P 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
phatase that is targeted specifically to Mid1 by ?4. Human birth
defects may result from environmental or genetic disruption of this
normalities, is associated with midline abnormalities such as cleft
lip, laryngeal cleft, heart defects, hypertelorism, hypospadias, im-
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
and?or 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
he Opitz G?BBB syndrome (OS), also known as the hypospa-
dias–dysphagia syndrome or telecanthus with associated ab-
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
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
yeast target of rapamycin (TOR)-signaling pathway (7–12). Yeast
Tap42 binds protein phosphatases Sit4 and Pph21?22 that corre-
spond to mammalian PP6 and PP2A, respectively (7), and binding
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-
and, therefore, might be cellular substrates of this form of phos-
phatase. Yeast two-hybrid screening with mouse ?4 as bait yielded
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: email@example.com.
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.
June 5, 2001 ?
vol. 98 ?
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 ? 106independent 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.Mid1full-lengthcDNAwasderivedfrom
c-Myc?green 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% CO2. Cells were grown to 50–60% 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-
conjugated Agarose (Santa Cruz ) and immunoblotting were done
as described previously (18).
Metabolic [32P] Labeling. COS7 cells in 60-mm dishes were trans-
supplemented with dialyzed newborn calf serum. Cells then were
labeled for 1.5 h in the same medium containing 1 mCi?ml
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 7.0?6 mM
EGTA?2.4 mM MgCl2) 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).
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
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
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
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
transfected with FLAG-tagged Mid1(58–180) with or without myc-tagged ?4.
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,
vector alone, FLAG-?4 (full-length), FLAG-?4(1–249), FLAG-?4(220–340), or
FLAG-?4 coprecipitated endogenous Mid1 and PP2A-C, detected by anti-FLAG,
anti-Mid1, and anti-PP2A-C immunoblotting.
Association of ?4 with Mid1 and PP2A. (a) Yeast two-hybrid analysis
Liu et al.
June 5, 2001 ?
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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-?4 could bind to
both endogenous Mid1 and the C subunit of PP2A. Although from
these results one cannot tell whether all three proteins, Mid1, ?4,
such complexes can assemble in living cells.
We mapped the regions in ?4 required for the binding to Mid1
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
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.
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).
www.pnas.org?cgi?doi?10.1073?pnas.111154698 Liu et al.
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 ?4 to a
region of residues 1–111 gave a protein that bound no PP2A C
subunit, indicating that a region between 111 and 249 of ?4 is
assignment of the phosphatase-binding site in ?4 (14). Our results
of ?4 most conserved between species. Distinct regions of ?4 bind
PP2A and Mid1, seemingly independent of one another.
Colocalization of ?4 and GFP-Mid1 with Microtubules. Recentstudies
have found that a Mid1-GFP fusion protein colocalized with
microtubules (4, 5). As expected, this GFP-Mid1 protein expressed
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
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
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
OS mutants of GFP-Mid1 associate with FLAG-?4. (a) COS7 cells were trans-
Liu et al.
June 5, 2001 ?
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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 and?or
the time of expression. Instead, GFP-Mid1 became distributed as
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
note that even when essentially all the GFP-Mid1 was accumulated
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
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
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.
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
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-
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
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-
(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
Phosphorylation–Dephosphorylation of Mid1.CouldMid1beaphos-
phoprotein, and, if so, does binding to the ?4-phosphatase reduce
required for Mid1 association with microtubules. (a) COS7 cells were cotrans-
Immunoprecipitation was performed by using anti-FLAG M2 affinity gel, and
COS7 cells were transfected as described and radiolabeled with [32P]orthophos-
phate for 90 min. GFP-Mid1 was immunoprecipitated with anti-GFP antibodies
and was subjected to PhosphorImager analysis after SDS?PAGE. 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.
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
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
Model for regulation of Mid1 association with microtubules by phos-
www.pnas.org?cgi?doi?10.1073?pnas.111154698 Liu et al.
its phosphorylation? We transfected COS7 cells to express GFP- Download full-text
Mid1 plus either empty FLAG vector or FLAG-?4. Anti-FLAG
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 with32Pi, and GFP-Mid1 protein was
recovered by anti-GFP immunoprecipitation. PhosphorImager
effect on the GFP-Mid1 expression level. Our interpretation of
promoting Mid1 dephosphorylation in living cells.
Only one site of phosphorylation is predicted in Mid1, based on
substrate for MAP kinase (http:??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).
Mid1 association with microtubules.
These data support a model (Fig. 5), with phosphorylation of
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
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
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 phosphorylation?dephosphorylation
mechanism. The potential ability of Mid1 to control cell behavior
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 of32P labeling in meta-
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
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
pathway to the regulation of microtubule stability and function.
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
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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