From the Department of Biology, West Virginia University,
Morgantown, West Virginia 26506-6057 and the
Department of Biochemistry, The Hebrew
University-Hadassah Medical School, Jerusalem 91120, Israel
Received for publication, July 7, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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Drosophila melanogaster
casein kinase II (DmCKII) is composed of catalytic ( Casein kinase II (CKII)1
is a ubiquitous protein kinase that is highly conserved among
eukaryotes (1, 2) and is capable of functioning as an oncogene in
mammals (3). CKII is composed of catalytic ( CKII preferentially phosphorylates Ser/Thr residues in an hyperacidic
context (10), although phosphorylation of Tyr has been documented in at
least one case, i.e. yeast Fpr3 (11). Analysis of the
phosphorylation of synthetic peptides suggests that the consensus site
for phosphorylation by CKII can best be described as
(S/T)(D/E)X(D/E) (10). Consistent with this, a number
of proteins critical for transcription, cell cycle regulation, and
signal transduction contain such a site(s) and are known to be
phosphorylated in vitro and in vivo (12).
Although CKII activity is inhibited in vitro by polyacidic
compounds, such as polyaspartate and polyglutamate (13), and stimulated
by polybasic compounds, such as polylysine and protamine (14), the
in vivo relevance of these observations is currently
unknown. Comparisons between recombinant monomeric Genetic analyses in budding and fission yeast have demonstrated
that the enzyme is essential for viability (19, 20). Studies utilizing
temperature-sensitive alleles of the In an attempt to better define the physiological role of CKII, we have
used the two-hybrid approach (24) to identify and characterize
physiological partners of the The studies described in this report demonstrate that in addition to
m7, DmCKII Construction of Two-hybrid Plasmids--
DNA corresponding to
amino acids 1-336 of DmCKII Gal4-based Yeast Two-hybrid Screening--
Screens were
conducted in the Gal4-based version of the two-hybrid system
(henceforth referred to as the Fields system) using the yeast strain
HF7C (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901,
leu2-3, 112, gal4-542, gal80-538,
LYS2::GAL1UAS-GAL1TATA-HIS3, URA3::GAL417mers(x3)-CyC1TATA-LacZ)
(45). HF7C expressing Gal4DB-DmCKII LexA-based Two-hybrid Interactions--
Explicit interactions
between DmCKII Purification of Glutathione S-Transferase (GST) Fusion
Proteins--
The construction of plasmids expressing E(spl) proteins
as C-terminal fusions with Schistosoma japonicum GST has
been described previously (38). Plasmids expressing GST-alone, GST-m7,
GST-m8, GST-m5, and GST-mC were transformed into E. coli
BL21(DE3) harboring the plasmid pT-TRX (gift of S. Ishii, Laboratory of
Molecular Genetics, Ibaraki, Japan). pT-TRX drives expression of
thioredoxin, which increases the solubility and functionality of
eukaryotic proteins expressed in E. coli (51). Cultures (100 ml) were grown in 2× YTA (47) containing 150 µg/ml ampicillin
and 15 µg/ml chloramphenicol to an A600
of 0.7 and induced with 1 mM
isopropyl- Purification of DmCKII as a Monomeric Catalytic Subunit and the
Phosphorylation of m5, m7, and m8 by CKII--
The reaction was
carried out at 25 °C in 50 mM Tris, pH 8.5, 100 mM NaCl, 10 mM MgCl2, 10 µM ATP, 5 µCi of [ Deletion Mapping and Site-directed Mutagenesis--
The
construction of variants of m7 lacking either the bHLH domain
(m7 In Vitro Interaction and Immunoblotting--
Two µg of
purified GST-alone or GST-m8 were mixed with 25 µl of
glutathione-Sepharose 4B and incubated overnight at 4 °C. The
Sepharose was pelleted by centrifugation for 1 min at 2000 × g, and the beads were washed once with 1.5 ml of wash buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 0.1% Triton X-100) to remove unbound GST fusion proteins. The washed Sepharose, containing the
immobilized GST fusion proteins, was then incubated with 100 ng of
purified Drosophila embryo CKII and incubated for 3 h
at 4 °C. The Sepharose was pelleted by centrifugation for 1 min at 2000 × g, and the supernatant was recovered as unbound
material. The pellets were washed two times for 5 min each time with
500 µl of wash buffer. Sepharose-bound (pellet) and unbound
(supernatant) fractions were resolved by SDS-polyacrylamide gel
electrophoresis and electrophoretically transferred to nitrocellulose
as described (53). DmCKII subunits were detected by Western blot
analysis using primary antibody against DmCKII (54) at a dilution of 1:1000 and secondary antibody (goat-anti-rabbit IgG coupled to alkaline
phosphatase, Bio-Rad) at a dilution of 1:3000. Immunoblots were
visualized using nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate (47).
Isolation of cDNAs Encoding m7--
The yeast strain HF7C
expressing Gal4DB-DmCKII
Sequencing revealed that the DmA51 cDNA encodes full-length m7 and
contains 78 and 228 base pairs of sequence, 5' to the initiation codon
(ATG) and 3' to the termination codon (TAA), respectively. This
cDNA, which does not contain any in-frame stop codons 5' to the
initiation codon, is identical to base pairs 172-1068 of a
4.4-kilobase genomic clone (Fig. 1B) that encodes the
m7 and m8 transcription units, each on a single uninterrupted exon
(55). The absence of a poly(A) tail in clone DmA51, combined with the presence of a single poly(A) addition signal in the corresponding gene
at position 1250 (Fig. 1B), suggests that the isolated
cDNA is not full-length with respect to its 3' untranslated region. In this regard, we have recently rescreened the Drosophila
cDNA library for DmCKII Interaction of DmCKII
The apparent specificity of DmCKII Conservation of a CKII Site in m5, m7, and m8--
As mentioned
above, all E(spl)C-derived proteins are structurally
conserved (32). However, the sequence alignment presented by Delidakis
and Artavanis-Tsakonas (32) emphasized conservation of the HLH domain,
helices III and IV (also known as Orange domain), a motif in the
vicinity of the C terminus with a high PEST score, and the WRPW motif
(Fig. 3A). We therefore
aligned the seven E(spl) proteins with emphasis on residues N-terminal
to the basic domain and those comprising the region from helix IV to
the C terminus (C-domain) to determine whether some structural features
were unique only to m5, m7, and m8. No sequences in the N terminus were
found that were conserved among and/or unique to these three proteins
(data not shown). On the other hand, analysis of the C-domain indicates
that only these three proteins contain a consensus site for
phosphorylation by CKII, 156SDNE in m5,
168SDNE in m7, and 159SDCD in m8, immediately
following the highly conserved sequence, (I/L)SP(V/A)SSGY (Fig.
3B), in a region that is characterized by a high PEST score
(32). Although PEST-rich sequences act as cis-acting signals that
regulate protein turnover (56) and have been suggested to be activated
via phosphorylation (see below), the role of this motif in m5/7/8 is
currently unknown. This conserved Ser in m5/7/8 conforms to the
requirement that it must contain an acidic residue at the
n+1 and n+3 positions to be a target for CKII
(10, 57, 58). It should be noted, that although mB also contains a site
for phosphorylation by CKII (195SEDE), it is neither
preceded by the (I/L)SP(V/A)SSGY sequence nor contained within its PEST
motif. Interestingly, the cytology and spatial organization of the
E(spl)C locus of Drosophila hydei exhibits an
extraordinary level of conservation relative to that of D. melanogaster (59). Because the DNA sequence of only D. hydei m8 is currently available, we have compared this protein with m5, m7, and m8 from D. melanogaster. Remarkably,
D. hydei m8 also contains the CKII site following the
(I/L)SP(V/A)SSGY sequence, both of which are contained within a region
with a high PEST score (see Fig. 3B). Given that the two
species, D. melanogaster and D. hydei, diverged
~60 million years ago (60), evolutionary principles would argue
against the notion that the conservation of the aforementioned motifs
is merely incidental. A more compelling case will become apparent once
the sequences encoding other E(spl) proteins of D. hydei
become available.
Phosphorylation of m5, m7 and m8 by Drosophila CKII--
One
question raised by the sequence alignment was whether the presence of
the consensus CKII site in m5, m7, and m8 correlates with their
phosphorylation. We have therefore subjected GST-alone, GST-m5, GST-m7,
GST-m8, and GST-mC, a noninteracting member, to phosphorylation using
two isoforms of CKII, i.e. the monomeric
We and others have previously demonstrated that polybasic compounds,
e.g. polylysine, overcome a down-regulation of the
holoenzyme that can be conferred by the
Is there any evidence, apart from our two-hybrid and phosphorylation
analysis, to suggest that m5/7/8 are more closely related to each other
than are other E(spl) proteins? We believe that molecular/genetic
analyses do, in fact, support this proposal. Using a bacteriophage
Interaction of m8 with DmCKII--
Although the available data
demonstrate that m5/7/8 interact with DmCKII and are phosphorylated by
it, they do not indicate whether these proteins are capable of direct
physical association. Analysis of complex formation between these
proteins in the developing Drosophila embryo is currently
precluded by the absence of antibodies that specifically recognize the
m8 protein (68), coupled with its restricted expression domains within
the neuroectoderm (33). As an alternative, we have assessed the ability
of recombinant bacterially expressed GST-m8 to form a physical complex
with CKII purified from Drosophila embryos. To this end,
GST-alone and GST-m8 were purified, immobilized on
glutathione-Sepharose beads, and tested for their ability to form a
complex with Drosophila embryo CKII. The presence of DmCKII
in the bead-bound (pellet) and unbound (supernatant) fractions was
assessed by Western blotting using an antisera that recognizes both
subunits ( Mapping the Site of Phosphorylation on m8--
We next sought to
define the site of phosphorylation. We therefore generated two variants
of m8 with substitutions of the conserved Ser in the CKII site,
i.e. m8S159A and m8S159D. The former is a
nonphosphorylatable variant, whereas the latter should mimic the
constitutively phosphorylated protein, in line with studies on ANTP
(27), HP1 (69), etc. GST-m8, GST-m8S159A, and GST-m8S159D fusion
proteins were purified and subjected to phosphorylation using DmCKII Interaction of m8S159A and m8S159D with DmCKII Implications of Phosphorylation of m5, m7, and m8--
The results
presented above raise the likely prospect that DmCKII interacts with
m5/7/8 when these proteins are in the nonphosphorylated state and that
the complexes dissociate upon phosphorylation. We obviously cannot
extrapolate the two-hybrid and biochemical results to the situation in
the epidermal precursors in the developing Drosophila embryo
with certainty. However, given the requirements of CKII for cell
cycle progression (21) and for checkpoint control (72), it is likely
that epidermal progenitors, which are expressing E(spl) proteins, also
contain CKII. A direct test of this proposal in the developing embryo
still remains a difficult task due to restricted expression of m5/7/8
and the absence of isoform-specific antibodies (see above). At a
functional level, our data indicate that interaction and/or
phosphorylation of m5/7/8 is unlikely to affect their DNA binding
properties (which require the basic region), their ability to
heterodimerize with proneural proteins (which requires the HLH domain),
or their ability to interact with Groucho (which requires the WRPW
motif). What function could then be ascribed to interaction and/or
phosphorylation? The structural and functional properties common to
m5/7/8, and by extension those in D. hydei m8, provide the
basis for a likely possibility. As mentioned above, all three proteins
contain a PEST-rich motif that harbors an invariant Ser residue that is
phosphorylated by CKII. In this regard, a mutation that removes
sequences encompassing the PEST-rich region and the resident CKII site
acts as a dominant-negative allele with regard to suppression of
bristle development (67). That this variant of m8 behaves as a
dominant-negative, rather than a loss-of-function (as one would have
predicted), suggests that the mutant protein might sequester endogenous
wild-type m8, and possibly m5 and m7 as well, thus leading to enhanced
neurogenesis. Thus, Giebel and Campos-Ortega (67) propose that this
region negatively regulates the activity of m8, a suggestion in line with its ability to homodimerize or heterodimerize with m5 and m7 (42,
43). These results and their interpretations are consistent with our
proposal that this region of m5/7/8 may influence the stability of
these proteins in vivo. In this regard, an interesting parallel has been identified, i.e. activation of the
morphogenic protein Dorsal in Drosophila and that of NF-
In summary, the data presented herein demonstrate that select members
of the E(spl)C, i.e. m5, m7, and m8, physically
interact with DmCKII and are phosphorylated by this enzyme at an
invariant Ser residue that is contained within a motif unique to these
three isoforms. The suggestion that these three proteins are more
functionally related (42, 43, 59, 68) and that the C-terminal domain of
m8 acts to negatively regulate function in vivo (67)
implicates the PEST motif and its resident CKII phosphorylation site.
We believe that the data presented strengthen our contention for the
presence of a new functional motif in these transcriptional repressors
and raise the possibility that CKII may regulate neurogenesis via
posttranslational modification of these proteins.
) and regulatory
(
) subunits associated as an
2
2
heterotetramer. Using the two-hybrid system, we have screened a
D. melanogaster embryo cDNA library for proteins that
interact with DmCKII
. One of the cDNAs isolated in this screen
encodes m7, a basic helix-loop-helix (bHLH)-type transcription factor encoded by the Enhancer of split complex
(E(spl)C), which regulates neurogenesis. m7 interacts with
DmCKII
but not with DmCKII
, suggesting that this interaction is
specific for the catalytic subunit of DmCKII. In addition to m7, we
demonstrate that DmCKII
also interacts with two other
E(spl)C-derived bHLH proteins, m5 and m8, but not with
other members, such as m3 and mC. Consistent with the specificity
observed for the interaction of DmCKII
with these bHLH proteins,
sequence alignment suggests that only m5, m7, and m8 contain a
consensus site for phosphorylation by CKII within a subdomain unique to
these three proteins. Accordingly, these three proteins are
phosphorylated by DmCKII
, as well as by the
2
2 holoenzyme purified from
Drosophila embryos. In line with the prediction of a single
consensus site for CKII, replacement of Ser159 of m8 with
either Ala or Asp abolishes phosphorylation, identifying this residue
as the site of phosphorylation. We also demonstrate that m8 forms a
direct physical complex with purified DmCKII, corroborating the
observed two-hybrid interaction between these proteins. Finally,
substitution of Ser159 of m8 with Ala attenuates
interaction with DmCKII
, whereas substitution with Asp abolishes the
interaction. These studies constitute the first demonstration
that DmCKII interacts with and phosphorylates m5, m7, and m8 and
suggest a biochemical and/or structural basis for the functional
equivalency of these bHLH proteins that is observed in the context of neurogenesis.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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) and regulatory (
)
subunits that combine to form an
2
2
holoenzyme. With the exceptions of Drosophila melanogaster (4), Caenorhabditis elegans (5), and
Schizosaccharomyces pombe (6), CKII from most eukaryotic
organisms contains two
subunits,
and
', that are encoded by
distinct genes. In contrast,
subunit heterogeneity has been
documented via protein microchemical approaches in Saccharomyces
cerevisiae (7) and via molecular/genetic approaches in
Arabidopsis thaliana (8) and D. melanogaster (9).
subunit and
native or reconstituted
2
2 holoenzyme
have revealed that the
subunit plays a complex role in regulating
the basal activity of the
subunit (15-17). Although the
subunit stimulates the activity of the monomeric
subunit ~5-fold
against most substrates, it down-regulates phosphorylation of a
select few proteins, notably calmodulin (14, 18), the actin
bundling protein, Sac6p,2 and
a novel Drosophila zinc finger protein,
ZFP35.3 The
subunit is
also subject to phosphorylation by the
subunit (4), but the
biological role of this reaction remains undefined.
subunits of yeast CKII
indicate a requirement of the enzyme for cell cycle progression in
G1 and G2/M (21), in the maintenance of
cytoskeletal architecture (22), and for cytokinesis (20). In contrast
to the two yeast models, analysis of DmCKII has been stymied,
principally due to the absence of mutations, even though the cDNAs
encoding DmCKII were the first to be isolated (23). However, two
complementary approaches that have recently been applied to DmCKII have
proven to be exceptionally useful for analyzing functions of this
kinase in a metazoan context. The first of these is the two-hybrid
system (24) that has been used to identify CKII interacting proteins, many of which appear to be potential substrates of this kinase. The
second is genetic analysis on these interacting proteins via targeted
misexpression of transgenes encoding nonphosphorylatable and
constitutively phosphorylated variants using the Gal4-UAS system (25),
followed by phenotypic analysis. Studies along these lines have
identified a novel regulatory (
') subunit of DmCKII (9), suggested
the presence of five alternative transcription start sites in the
CKII
gene (26), and demonstrated an interaction of the
homeobox protein Antennapedia, ANTP, with DmCKII
(27). In the
case of ANTP, phosphorylation by DmCKII appears necessary for
restricting its activity during embryogenesis. A somewhat similar
situation exists for another homeobox protein, Engrailed, which also
appears to be regulated by CKII-mediated phosphorylation (28). In
addition, the segment polarity protein Dishevelled, a component
of the wingless/Wnt signaling pathway (29), is a target for CKII, and
both proteins exist as a complex in vivo (30). Collectively,
these results suggest that CKII plays crucial roles in embryonic
development as well as in cellular differentiation.
subunit of DmCKII (DmCKII
). One of
the proteins identified in this screen is m7, a bHLH-type transcription
factor derived from the neurogenic locus E(spl)C (31). The
E(spl)C encodes the structurally and functionally similar
bHLH proteins mC (also known as m
), mB (also known as m
),
mA (also known as m
), m3, m5, m7, and m8 (32, 33) and is epistatic
to other neurogenic loci, such as Notch, Delta,
etc. (34-36). The segregation of neural and epidermal lineages during development is determined by cell-cell communications that involve two
interacting sets of genes: the neurogenic genes mediate signals between
adjacent cells, and the proneural genes promote neural development.
This nomenclature is explained by the fact that the neurogenic genes
are named for their loss-of-function phenotype, whereas the proneural
genes are named for their normal function. In a process termed
"lateral inhibition," Delta provides the inhibitory signal that is
received by Notch. The strength of this signal in the target cell
determines functional predominance of the products of either the
achaete-scute complex or the E(spl)C.
Predominance of the former leads to neurogenesis, whereas that of the
latter leads to inhibition of neurogenesis, i.e.
epidermogenesis (37). Proteins of the E(spl)C form
heterodimers with Groucho (38, 39), a nuclear protein that contains
WD40 repeats (40). This interaction occurs via a C-terminal
tetrapeptide, WRPW, that is invariant among all E(spl)C
members, as well as in Hairy and Deadpan, which regulate segmentation
and sex determination, respectively (38). E(spl)C-derived
bHLH proteins have been proposed to inhibit neurogenesis via
transcriptional repression of proneural genes by binding the N-box
sequence, CACNAG (41), as well as by sequestering the proneural
proteins themselves (42, 43).
also interacts with two other E(spl) proteins, m5 and m8,
and that all three proteins are phosphorylated by DmCKII in
vitro. Protein-protein interaction analysis demonstrates a direct
physical association between m8 and Drosophila embryo CKII. Using site-directed mutagenesis in combination with phosphorylation and
two-hybrid analyses, we have mapped the site of phosphorylation, and we
demonstrate that replacement of the phosphoacceptor Ser with Ala (a
nonphosphorylatable residue) attenuates interaction of m8 with CKII,
whereas substitution with Asp (which mimics the constitutively
phosphorylated protein) abolishes the interaction. These studies
demonstrate that the bHLH proteins m5, m7, and m8 are new physiological
partners and substrates of DmCKII.
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ABSTRACT
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RESULTS AND DISCUSSION
REFERENCES
and amino acids 1-215 of DmCKII
was
amplified by polymerase chain reaction using primers containing two
terminal 5' bases, a restriction site, and 20 bases of exact homology
to the start and stop codon regions. The polymerase chain reaction
products were subcloned into the plasmids pGBT9 and pGAD424 (gift of S. Fields, University of Washington) and completely sequenced using the
Prism Dye Terminator Cycle sequencing kit (Applied Biosystems). The
resulting plasmids express DmCKII
and DmCKII
as C-terminal
fusions with the DNA binding (DB) domain, and activation domain
(AD) of S. cerevisiae Gal4 (44), respectively.
(the bait) was used to screen a
3-18-h D. melanogaster embryo two-hybrid cDNA library
(gift of S. J. Elledge, Baylor College of Medicine). This library
is contained in the plasmid pACT, which expresses cDNA-derived
proteins as C-terminal fusions with Gal4AD (46). A total of 2 × 106 transformants were plated on glucose dropout medium
lacking tryptophan, leucine, and histidine (47), and colonies
exhibiting rapid growth were counterscreened for expression of
LacZ (48). Of the 45 His+ colonies, 15 tested
positive for LacZ and were therefore chosen for further
analysis. The library plasmids containing the yeast LEU2
gene were selectively recovered via complementation of the leucine
auxotrophy of Escherichia coli HB101 (47). The isolated plasmids were subsequently used to retransform HF7C expressing Gal4DB-alone, GAL4DB-DmCKII
and GAL4DB-DmCKII
. Those
cDNAs that induced expression of HIS3 and
LacZ only in response to GAL4DB-DmCKII
, i.e. a
bait-specific manner, were identified by sequencing their 5'- and
3'-ends using the primers 5'-ATACCACTACAATGGATGATG-3' and
5'-ACAGTTGAAGTGAACTTGCG-3', respectively. All novel cDNAs were
completely sequenced using custom primers as described above. One of
these cDNAs, DmA51, which encodes the bHLH protein m7, is the
subject of this study, whereas others will be reported elsewhere.
and E(spl) proteins were studied in the LexA-based
version of the two-hybrid system (49) that was developed in the
laboratory of Roger Brent (henceforth referred to as the Brent system).
In the Brent system, proteins to be tested for interaction are
expressed as fusions with the DNA binding domain of the bacterial
repressor, LexA, and the activation domain of protein B42. The yeast
strain used for these studies was EGY048 (MATa, trp1, his3, ura3,
leu2), which harbors a single chromosomally integrated copy of the
yeast LEU2 gene under the control of six LexA
operators, and a high copy plasmid, pSH18-34, which expresses E. coli LacZ under the control of eight LexA operators (50). Therefore, expression of the two reporter genes, LEU2 and LacZ, is induced when the interacting complex is
tethered to the LexA-operators. Additionally, expression of
the AD fusion protein is under control of a GAL-promoter. As
a result, reporter gene expression, in an AD fusion
protein-dependent manner, is only observed when cells are
grown in media containing galactose, but not glucose, as the sole
carbon source. Yeast EGY048 containing plasmid pSH18-34 was transformed
with a plasmid expressing the B42-derived AD-alone (49) or AD-DmCKII
fusion protein using lithium acetate (47). A single transformant was
selected and subsequently retransformed with plasmids expressing
LexA-m7, LexA-m8, LexA-m5, LexA-m3, and LexA-mC (38). Three independent
transformants were tested for induction of the LEU2 gene on
glucose- and galactose-dropout medium lacking leucine (47) at 29 °C
for 4 days. In parallel, cultures were analyzed in triplicate for
-galactosidase (LacZ) activity using a filter, as
well as a solution-based assay (48).
-D-thiogalactoside for 3 h at
30 °C with vigorous shaking. All subsequent steps were conducted at
4 °C. Cells were harvested, resuspended in 8 ml of phosphate-buffered saline containing 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM EDTA, and 0.2%
2-mercaptoethanol and lysed by sonication. Phase contrast microscopy
was used to ensure greater than 95% cell lysis. Triton X-100 was added
to a final concentration of 1% and mixed for 3 h at 4 °C.
Insoluble material was removed by centrifugation, and the supernatant
was passed twice through a column containing 1 ml of
glutathione-Sepharose 4B (Amersham Pharmacia Biotech). The column was
washed with 10 bed volumes of phosphate-buffered saline, and bound
protein was eluted with 5 ml of 100 mM reduced glutathione
in 50 mM Tris, pH 8.0. The eluted protein was concentrated and exchanged into storage buffer (10 mM Tris, pH 8.0, 0.5 mM EDTA, 5% glycerol, 150 mM NaCl, and 0.1 mM phenylmethylsulfonyl fluoride) using a Biomax-10K
centrifugal filter device (Millipore). The purity of the fusion
proteins was determined by SDS-polyacrylamide gel
electrophoresis essentially as described (52), and their concentration was estimated from Coomassie Blue-stained gels relative to known standards.
2
2 Holoenzyme--
The monomeric
subunit of DmCKII was purified to homogeneity from an S. cerevisiae expression system as described (15), and the tetrameric
holoenzyme was purified from embryos according to Glover et
al. (4), with modifications that will be described elsewhere. The
Vmax of the
subunit monomer is 0.4 µmol/min/mg, whereas that of the holoenzyme is 1.6 µmol/min/mg,
using partially hydrolyzed and dephosphorylated casein (Sigma) as a
substrate. These values are similar to those reported earlier (14).
-32P]ATP, and ~2
µg of various GST-E(spl) fusion proteins in a total volume of 40 µl. The reaction was initiated with 5 µl of the enzyme (either as
the
subunit monomer or the holoenzyme) at a concentration of 8 µg/ml in 20 mM Tris, pH 8.0, 0.5 mM EDTA, 200 mM NaCl, 10% glycerol, 0.5 mM dithiothreitol,
and 0.05% Triton X-100. To study the effects of
poly(DL)lysine on phosphorylation, reactions were supplemented to a final concentration of 100 µg/ml. The reactions were terminated with 10 µl of 5× sample buffer (312 mM
Tris-Cl, pH 6.8, 10% SDS, 25% 2-mercaptoethanol, and 40% glycerol),
and boiled for 5 min. Samples were separated by SDS-polyacrylamide gel
electrophoresis and stained with Coomassie Blue, and the destained gels
were exposed to Kodak XAR-5 film at room temperature.
bHLH), or the WRPW motif (m7
WRPW) has been described previously (38). Two variants for mapping of the phosphorylation site
were made in the cDNA encoding m8 using the Quick-Change site-directed mutagenesis kit (Stratagene). These are m8S159A (which replaces Ser159 with Ala), and m8S159D (which
replaces Ser159 with Asp). The two complementary primer
sets used for the former variant were
5'-CCGGATATCACGCCGACTGCGACAGC-3' and
5'-GCTGTCGCAGTCGGCGTGATATCCGG-3', and those for the
latter variant were 5'-CCGGATATCACGACGACTGCGACAGC-3' and
5'-GCTGTCGCAGTCGTCGTGATATCCGG-3', respectively. The
underlined bases correspond to those substituting Ser159
with either Ala or Asp. A plasmid containing the complete open reading
frame encoding m8 was subjected to 17 cycles of polymerase chain
reaction using the primer sets described above, and the polymerase
chain reaction product was digested with the enzyme DpnI to eliminate
the nonmutant plasmid that was used as a template. The reaction mixture
was used to transform E. coli DH5
, and the cDNA from
a representative transformant was completely sequenced on both strands
using custom primers. Subsequently, the cDNAs encoding m8S159A and
m8S159D were subcloned into the EcoRI-BamHI sites
of the vectors, pZEX, for expression and purification of GST fusion
proteins (38), and pEG202, for expression as LexA fusions for
two-hybrid analysis (49). Phosphorylation of wild-type m8 and the two
variants, and their interactions with DmCKII
in the Brent system
were conducted as described above.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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as a bait was used to screen a D. melanogaster embryo two-hybrid cDNA library. From ~2 × 106 transformants, 15 clones that activated transcription
of HIS3 and LacZ were recovered. All 15 clones
induced the two reporter genes only when cotransformed with DmCKII
(data not shown). Sequencing of the cDNAs revealed that seven of
the clones encode DmCKII
(26), one encodes DmCKII
(9), two encode
DmCKII
' (a novel isoform of the
subunit (9)), one (DmA51)
encodes m7, and the rest encode novel proteins that will be described
elsewhere. The library plasmid was recovered from yeast clone DmA51 and
retested for interaction against various bait constructs. As shown in
Fig. 1A, induction of
HIS3 and LacZ was observed only when yeast HF7C coexpressed Gal4AD-m7 with Gal4DB-DmCKII
. On the other hand, neither
reporter gene was induced when HF7C was transformed with Gal4AD-m7 by
itself or in combination with a plasmid encoding either Gal4DB-alone or
Gal4DB-DmCKII
, suggesting that m7 interacts specifically with the
catalytic subunit of DmCKII. The inability of DmCKII
to interact
with m7 is not due to a lack of expression of the former protein
because this construct is expressed in yeast and displays a strong
interaction with DmCKII
(9).
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Fig. 1.
Isolation of m7 and interaction with DmCKII
subunits. A, S. cerevisiae strain HF7C was
transformed with plasmids expressing the indicated fusions with GAL4DB
or GAL4AD. Untransformed HF7C was used as a control; a dashed
line indicates the absence of a plasmid, and vector
indicates the presence of a plasmid expressing Gal4DB-alone. Following
growth in minimal medium lacking leucine and tryptophan, induction of
the two-hybrid reporter genes, HIS3 and LacZ, was
assayed as described under "Experimental Procedures."
-Galactosidase (LacZ) activity is expressed in Miller
units, and the mean and S.D. of three replicate assays are shown.
B, the start and end points of the 897-base pair cDNA
encoded by two-hybrid clone DmA51 (hatched box) are
shown relative to sequences contained within a 4462-base pair
genomic clone (GenBankTM accession number X16553)
(open box) that encodes m7 and m8. The open reading
frame encompassing m7 (m7 ORF) is contained within a single
uninterrupted exon (shaded box). The locations of the
transcription start site and the polyadenylation signal
(AATAAA) are indicated.
-interacting proteins and have isolated
two additional clones, DmA002 and DmA130, that also encode m7. Our isolation of multiple cDNAs encoding m7 from two independent
two-hybrid screens strengthens the likelihood of the relevance of its
interaction with DmCKII
. Apart from length heterogeneity with
respect to the DmA51 cDNA, the DmA002 and DmA130 sequences are
identical to the corresponding region of the m7 transcription unit and
display no polymorphisms (data not shown).
with E(spl)C-derived bHLH
Proteins--
The observed interaction between DmCKII
and m7 was
surprising, as there was no previous indication that m7 is regulated by phosphorylation or that CKII is involved in neurogenesis. Given the
structural similarity of all E(spl) proteins (32), we were interested
in determining whether DmCKII
also interacts with other members
derived from this locus. For this purpose, we made use of the Brent
system (see under "Experimental Procedures") to remain consistent
with the analysis of Paroush et al. (38), who have
convincingly demonstrated the interaction of E(spl) proteins with
Groucho. We therefore transformed yeast EGY048 with plasmids expressing
the AD-alone or the AD-DmCKII
fusion protein, and the resulting
strains were retransformed with plasmids expressing various LexA-E(spl)
fusion proteins (38). As shown in Fig. 2, induction of LacZ was specifically observed when cells
coexpressed DmCKII
with m5, m7, or m8. The levels of LacZ
induced with m3 or mC were similar to those obtained with LexA-alone,
suggesting that the DmCKII
-m5/7/8 interactions are not mediated via
the LexA domain. Furthermore, no significant reporter gene expression was observed when m5/7/8 were tested against the AD-alone, indicating specificity with regard to DmCKII
. The inability of m3 and mC to
interact with DmCKII
is not due to attenuated/lack of expression, as
these constructs display robust interactions with Groucho that are
equivalent to those observed with m5, m7, and m8 (38). The higher
levels of LacZ activity observed for mC in combination with
the AD-alone and its "silencing" upon expression of AD-DmCKII
are consistent with our observation that the unfused AD, in a limited
number of cases, confers basal transcription of the reporter genes that
is abolished upon expression as a fusion
protein.4 That DmCKII
-m5,
-m7, and -m8 interactions display a higher LacZ activity
(Fig. 2) than does DmCKII
-DmCKII
(Fig. 1A) does not imply that the former protein pairs interact with a higher affinity. As
outlined under "Experimental Procedures," this is a reflection of a
high copy/affinity LacZ reporter used in the Brent system. In addition, no induction of LacZ activity was observed when
transformants were grown in rich glucose medium (data not
shown), suggesting that reporter gene expression was dependent on
presence of the AD-DmCKII
fusion protein. Identical results were
observed for the second reporter gene, LEU2 (data not
shown). It should be noted that the two-hybrid interaction between
DmCKII
and m7 was originally detected using the former protein as a
Gal4DB fusion and the latter as a Gal4AD fusion (Fig. 1A),
whereas the explicit testing involved the inverse orientation,
i.e. m7 as a LexA fusion and DmCKII
as an AD fusion (Fig.
2). These results demonstrate that the interactions are neither
orientation-specific, as is the case of the interaction of DmCKII
with DmCKII
(9), nor dependent on a specific version of the
two-hybrid system.
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Fig. 2.
Interaction of DmCKII
with E(spl)C-derived bHLH proteins.
S. cerevisiae strain EGY048 harboring the
LacZ-expression plasmid, pSH18-34, was transformed with
plasmids expressing the indicated LexA-bHLH fusion proteins in
combination with either the activation domain alone (hatched
bars) or the AD-DmCKII
fusion protein (shaded bars).
Transformants were grown in rich galactose medium, and the
levels of LacZ expressed were determined as described (48).
-Galactosidase (LacZ) activity is expressed in Miller
units, and the data shown are the average of three independent
experiments.
for m5, m7, and m8 but not for
other members tested (m3 and mC) was surprising given that these
proteins are structurally conserved (32). Of particular note are two
motifs, the highly conserved HLH domain, which mediates heterodimerization with proneural proteins such as Ac, Da, and l'sc
(42), and the invariant WRPW motif (32), which mediates interaction
with Groucho (38). We found, however, that variants of m7 lacking
either of these motifs interact as effectively with DmCKII
, as does
the wild-type protein (data not shown). These results are consistent
with our inability to detect interactions of DmCKII
with m3 and mC
(both of which contain the HLH and WRPW motifs) and suggest that the
interaction domain lies elsewhere in m5/7/8.
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Fig. 3.
Functional motifs common to proteins of the
E(spl)C and alignment of the sequences comprising
their C-terminal regions. A, schematic representation
of the functional motifs contained in E(spl) proteins are shown
boxed: checkerboard box, basic region; wavy
box, HLH domain; diamond shaded box, Orange domain,
which is predicted to form -helices III and IV; shaded
box, PEST-rich region; hatched box, the CKII site in
m5, m7, m8, and D. hydei m8 (Dh-m8); black box,
the C-terminal tetrapeptide WRPW. B, the amino acid
sequences of D. melanogaster E(spl) proteins and that of
D. hydei m8 (Dh-m8) were aligned using the Multiple Sequence
Alignment algorithm (Baylor College of Medicine), and aligned sequences
encompassing the C-domain are shown. The sequences shown and their
GenBankTM accession numbers are as follows: m5, X16552; m7,
X16553; m8, X16553; m3, M96165; mA, X67047; mB, X67049; mC, X67048; and
Dh-m8, X71662. The invariant WRPW tetrapeptide is shown in
boldface italics, the PEST motif is
underlined, the consensus site for phosphorylation by CKII
is shown in boldface and enclosed in a shaded box, with an
arrow indicating the likely phosphoacceptor, the
(I/L)SP(V/A)SSGY motif is shown as an open box, and the
asterisks at the beginning of the m7 sequence indicate that
the PEST motif extends N-terminal to the residues shown in the
alignment.
subunit
purified from a yeast expression system (15), and the
2
2 holoenzyme purified from embryos (4).
The former isoform mimics the two-hybrid analysis (Fig. 2), whereas the
latter mimics the in vivo environment. The results
demonstrate that m5, m7, and m8 are phosphorylated by both isoforms of
CKII (Fig. 4, B and E,
lanes 2-4) and corroborate their observed two-hybrid interaction with DmCKII
. No phosphorylation of either GST or GST-mC (Fig. 4,
B and E, lanes 1 and 5) was observed
with either enzyme isoform, demonstrating the absence of
phosphorylation of the affinity tag used for purification and
suggesting that phosphorylation is specific only to those E(spl)
proteins that also exhibit a two-hybrid interaction with DmCKII
. At
a quantitative level, however, the rates of phosphorylation of the
three E(spl) proteins are different for both enzyme isoforms, such that
m5 > m7 = m8 (compare lanes 2, 3, and
4 in Fig. 4, B and E). What mechanism
can account for the observed differences? Detailed kinetic analysis of
CKII suggests that whereas DmCKII
and the holoenzyme display
virtually identical km values for the protein
substrate, the Kcat can differ 5-50-fold in a substrate-dependent manner (14). Furthermore, studies with
peptides suggest that whereas the acidic residues at n+1 and
n+3 are absolutely required for phosphorylation, additional
acidic residues C-terminal to the n+3 position further
increase the Kcat with marginal effects on the
km (57, 61). These criteria, therefore, make it
possible to predict the relative rates of phosphorylation of m5/7/8. In
this regard, although m7 and m8 fit the consensus, m5 is probably the
best because it contains an additional Asp at the n+4
position (Fig. 3B). The rank order for phosphorylation is,
therefore, predicted to be m5 > m7 = m8. The analysis
presented here essentially reflects this prediction. Because the gel
analysis described here inherently reflects a semiquantitative
assessment of phosphorylation, kinetic analysis will be necessary to
determine whether the observed differences in phosphorylation of m5
versus m7/8 are due to differing catalytic efficiencies
(Kcat/km). That CKII
interacts with and phosphorylates these proteins is consistent with the
observation that this kinase has been found to exist in a complex with
some of its in vivo substrates, such as Topoisomerase II
(62), HSP90 (63), ANTP (27), and Dishevelled (30), to name a few.
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Fig. 4.
Phosphorylation of m5, m7, and m8 by
DmCKII. The indicated GST fusion proteins were purified and
subjected to phosphorylation using the monomeric subunit
(DmCKII
) and the
2
2 holoenzyme from
Drosophila embryos. A representative gel stained with
Coomassie Blue shows the amount and purity of the various GST fusion
proteins that were phosphorylated with either DmCKII
(A)
or the holoenzyme (D). Proteins were phosphorylated in
either the absence (B and E) or in the presence
(C and F) of 100 µg/ml
poly(DL)lysine. Samples were electrophoresed in 12%
SDS-polyacrylamide gels, stained with Coomassie Blue, and
autoradiographed (B, C, E, and F).
Arrows in A and D indicate the
mobilities of the full-length fusion proteins.
subunit of CKII for some
substrates (14, 64). We were therefore interested in determining
whether the limited phosphorylation of m7 and m8, relative to that of m5, might be sensitive to polylysine addition. The results suggest that
phosphorylation of m5 is virtually unaffected by this compound when
tested with either DmCKII
(compare Fig. 4B, lane 2, to
Fig. 4C, lane 2) or DmCKII holoenzyme (compare Fig.
4E, lane 2, to Fig. 4F, lane 2). On the other
hand, phosphorylation of both m7 and m8 is stimulated by polylysine
addition (compare lanes 3 and 4 in Fig.
4B versus 4C and 4E
versus 4F). These results are consistent with
previous analysis indicating that phosphorylation of substrates with
optimal sites (such as the RII subunit of cAMP-dependent protein kinase) responds modestly, if at all, to polylysine, whereas those that satisfy the minimal requirements of CKII (such as
calmodulin) are more responsive (14). This stimulation by polylysine is not a reflection of promiscuous phosphorylation, because DmCKII
or
the holoenzyme do not phosphorylate GST or GST-mC (lanes
1 and 5 in Fig. 4, C and F) in
the presence of polylysine. However, no cellular protein that can mimic
the "polylysine effect" with respect to CKII has so far been
identified, unlike the case with Ras, which mediates the
polylysine-dependent phosphorylation of calmodulin by the
insulin-receptor kinase (65).
-based system to detect protein-protein interactions, Gigliani
et al. (43) suggest that m5, m7, and m8 are the most closely
related. Although yeast two-hybrid analysis conducted by Alifragis
et al. (42) essentially reiterates the closest similarity
between m5 and m8, they suggest, however, that m7 be clustered along
with mA and mB, a proposal at odds with their own genetic analysis (see
below). Because E(spl) proteins homo/heterodimerize and interact with
proneural proteins as well, in vivo associations between
these proteins is needed to clarify the differences, if any, with
regards to m7. Furthermore, and perhaps the most persuasive, is genetic
analysis demonstrating that the severity of suppression of bristle
development, i.e. neurogenesis, closely correlates with
ectopic expression of only m7 and m8 (66). A similar analysis with m5
was, however, precluded even with two copies of the transgene, leading
the authors to conclude that, of the seven E(spl) proteins, m5
is probably most inactive/unstable (66). Northern or Western
analysis on the m5 transgenics will be necessary to clarify whether
this is indeed the case. Given the functional equivalency of m5/7/8 in
neurogenesis, we were specifically interested in determining the
mechanism by which these proteins interact with DmCKII. We have
deferred conducting parallel, and thus redundant, analysis on all three
proteins and have selected m8 for these additional studies. This choice
was based on our eventual goal of analyzing the significance of this interaction in transgenic flies and to remain consistent with genetic
analysis on this protein (see below and Ref. 67) via the GAL4-UAS
system (25).
and
) of DmCKII (54). As expected, incubation of the
GST beads with DmCKII did not result in any immunoreactive material in
the pellet fraction, indicating the absence of an interaction (Fig.
5, compare lanes P and
S). On the other hand, incubation of GST-m8 beads with
DmCKII resulted in the presence of immunoreactive material in the
pellet fraction (Fig. 5, compare lanes P and S),
demonstrating that these two proteins form a physical complex. These
results suggest that the two-hybrid interaction of DmCKII with m8 is
direct and is unlikely to be mediated by the recruitment of yeast
proteins.
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Fig. 5.
Interaction of DmCKII with m8.
Bacterially expressed GST fusion proteins immobilized on
glutathione-Sepharose beads were incubated with Drosophila
embryo holoenzyme. The beads were separated from the unbound material
as described under "Experimental Procedures," and the bead-bound
(P, pellet) and the unbound (S, supernatant)
samples were examined by Western blotting using antisera raised against
Drosophila embryo CKII. The arrows indicate the
immunoreactive bands corresponding to the and
subunits of
DmCKII.
and the holoenzyme. The results demonstrate that GST-m8 is
phosphorylated by the holoenzyme and the
subunit (Fig.
6B, lanes 1 and 4).
On the other hand, neither GST-m8S159A (Fig. 6B, lanes 2 and
5) nor GST-m8S159D (Fig. 6B, lanes 3 and
6) are substrates of the two enzyme isoforms. This result
strongly suggests that CKII phosphorylation of m8 occurs at
Ser159, and by corollary the site of phosphorylation on m7
and m5 is most likely to be Ser168 and Ser156,
respectively (see Fig. 3B). We consider it unlikely that
GST-m8S159A and GST-m8S159D are partially clipped leading to abolished
phosphorylation, because, relative to GST-m8, neither protein exhibits
altered mobility in SDS-polyacrylamide gels (Fig. 6A,
compare lane 1 with lanes 2 and 3). In
addition, the inability of the two variants to be phosphorylated by
CKII suggest that m8 contains a single site for phosphorylation by
CKII, thus corroborating our sequence-based prediction (see Fig.
3B). The ability of m8 to be phosphorylated by DmCKII
and
the holoenzyme at the identical residue is also consistent with our
contention that the substrate specificity of this enzyme is intrinsic
to the
subunit (14).
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Fig. 6.
Mapping of the CKII phosphorylation site on
m8. A and B, the indicated GST fusion
proteins were purified and subjected to phosphorylation using the
2
2 holoenzyme from Drosophila
embryos (lanes 1-3) and the monomeric
subunit,
DmCKII
(lanes 4-6). Lanes 1 and 4, wild-type m8; lanes 2 and 5, m8S159A; lanes
3 and 6, m8S159D. Samples were electrophoresed in 12%
SDS-polyacrylamide gels, stained with Coomassie Blue (A),
and autoradiographed (B). The position of the GST fusion
proteins is indicated by the arrow.
--
We were
interested in determining whether phosphorylation of m8 affects its
interaction with CKII. We, therefore, determined the interaction of
DmCKII
with m8S159A and m8S159D, relative to wild-type m8. As shown
in Fig. 7, replacement of
Ser159 with Ala decreased interaction by ~50%, whereas
replacement with Asp abolished the interaction. These results suggest
that the interaction of DmCKII
with m8 appears analogous to that of
an enzyme with its substrate and is in line with the interactions of
the protein kinase, Snf1, with its substrate, Snf4 (70). Our
interpretation of the results is, however, complicated by the fact that
phosphorylation of m8 appears to disrupt the complex and may affect
protein stability as well (see below). The former possibility is
likely, given that DmCKII
is catalytically active when expressed in
yeast (15), and suggests that the strength of the two-hybrid
interaction observed between DmCKII
and m5/7/8 may, in fact,
represent an underestimate. The likelihood of the latter possibility is
difficult to predict, at least in the context of the yeast system used
in this study, given that two-hybrid interaction of m8S159D with
Groucho appears identical to that observed for wild-type
m8.4 These results suggest that accessory proteins, perhaps
lacking in yeast, may be necessary for affecting stability of m8S159D in Drosophila. In addition, although D. melanogaster m5/7/8 and D. hydei m8 contain the
conserved sequence, (L/I)SP(V/A)SSGY, flanking the phosphorylation site
(see Fig. 3B), it is presently unknown whether these
residues contribute binding energy in addition to that attributable to
the CKII site. Studies with Snf1/Snf4 have, in fact, demonstrated the
involvement of flanking residues in mediating interactions (71).
Further analysis will be needed to determine the intrinsic affinities
(Ka) of these bHLH proteins for DmCKII, rather than
those (km) inferred by kinetic analysis.
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Fig. 7.
Interaction of DmCKII
with m8, m8S159A, and m8S159D. S. cerevisiae
strain EGY048 harboring the LacZ-expression plasmid,
pSH18-34, was transformed with plasmids expressing the indicated LexA
fusion proteins in combination with either the activation domain alone
(hatched bars) or the AD-DmCKII
fusion protein
(shaded bars). Transformants were grown in rich galactose
media, and the levels of LacZ expressed were determined as
described (48).
-Galactosidase (LacZ) activity is
expressed in Miller units, and the data shown are the average of three
independent experiments.
B
in humans. Dorsoventral patterning in the Drosophila embryo
involves the activities of a transcription factor, Dorsal, and its
inhibitor, Cactus (73). Upon receiving the inductive signal, Cactus
appears to be phosphorylated by CKII within a motif with a high PEST
score (74) and undergoes degradation, thus allowing Dorsal to
translocate to the nucleus. A mechanistically similar situation appears
to regulate the NF-
B/C-Rel family of transcription factors in humans
(75, 76). A collective theme that emerges from these studies is that
phosphorylation, in at least a restricted class of proteins, regulates
protein stability via activation of PEST motifs. Our studies further
implicate the PEST motif in m5, m7, and m8 as a target for regulation
via CKII-mediated phosphorylation. Future studies employing expression of epitope-tagged m8 and its nonphosphorylatable and/or constitutively phosphorylated variants in transgenic flies will be needed to clarify
the role of this motif. If these studies indicate this to be the
case, we predict that the m8S159A variant would exhibit a longer
half-life in vivo, thus leading to its predominance over the
proneural proteins and therefore to an inhibition of neurogenesis. If
so, the m8S159D variant may exhibit a shorter half-life in vivo, thus preventing antagonism of the proneural proteins.
Experiments to address the role of phosphorylation on protein turnover
and effects on neurogenesis, via the transgenic route (67), are currently under way. In an interesting twist, Alifragis et
al. (42) report that a mutation in the proneural protein, Sc, that replaces Ser340 with Asp, abolishes its interaction with
m3. It should be noted that the sequences flanking Ser340
(DYIS340LWQEQ) do not conform to the consensus
for CKII or to that of other Ser/Thr protein kinases with defined
substrate specificities. Although their data (42) are conjectural, when
they are taken together with our results, it appears that neurogenic as
well as proneural proteins may be regulated by phosphorylation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Claiborne Glover and David Ish-Horowicz for providing valuable resources and Clifton Bishop and Philip Keeting for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by American Cancer Society Grant RPG-9918901-DDC (to A. P. B.) and by grants from the Israel Cancer Research Fund and the Jan M. and Eugenia Krol Charitable Foundation (to Z. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biology, P. O. Box 6057, Brooks Hall, West Virginia University, Morgantown, WV 26506-6057. Tel.: 304-293-5201 ext. 2533; Fax: 304-293-6363; E-mail: Abidwai@wvu.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M005996201
2 A. P. Bidwai, unpublished data.
3 M. Kalive, R. L. Trott, and A. P. Bidwai, unpublished data.
4 R. L. Trott and A. P. Bidwai, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CKII, casein kinase II; HLH, helix-loop-helix; bHLH, basic HLH; E(spl), Enhancer of split; E(spl)C, Enhancer of split complex; Dm, D. melanogaster; DB, DNA binding; AD, activation domain; GST, glutathione S-transferase; SDS, sodium dodecyl sulfate.
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