From the Department of Medicine, University of
Washington, Seattle, Washington 98195, the
German Cancer
Research Center, D-69120 Heidelberg, Germany, and the
§ Department of Gastroenterology at the Maria
Sklodowska-Curie Memorial Cancer Center and Institute of Oncology,
02-781 Warsaw, Poland
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ABSTRACT |
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The heterogeneous nuclear ribonucleoprotein
(hnRNP) K protein recruits a diversity of molecular partners that are
involved in signal transduction, transcription, RNA processing, and
translation. K protein is phosphorylated in vivo and
in vitro by inducible kinase(s) and contains several
potential sites for protein kinase C (PKC) phosphorylation. In this
study we show that K protein is phosphorylated in vitro by
PKC The hnRNP1 K protein has
a diverse repertoire of molecular partners that are involved in signal
transduction and gene expression. K protein binds with RNA,
single-stranded, and double-stranded DNA, and it associates with a
number of transcriptional activators and repressors, including
TATA-binding protein (1). K protein also interacts with tyrosine (2)
and serine/threonine kinases (3-5) as well as the proto-oncoprotein
Vav (3, 6, 7). The diverse molecular interaction of K protein may
account for the observations that K protein can both increase (8, 9) and decrease (10-12) gene transcription. For example, on one hand K
protein synergies with TATA-binding protein to increase transcription from the c-myc promoter CT element (8), whereas on the other it represses C/EBP K protein is made up of modular domains that bind different molecular
partners. For example, the three KH domains are thought to mediate
nucleic acid binding (19, 20), whereas the two clusters of SH3-binding
sites recruit the proto-oncoprotein Vav (3) and the Src-class of
tyrosine kinases (2). The latter sites are contained within the same
region that binds several transcriptional repressors, such as Zik1
(21), as well as the global regulator of anterior-posterior patterning,
Eed (22). This module is adjacent to a nuclear shuttling domain, KNS
(15), that may mediate direct coupling of K protein to a bona fide
nuclear/cytoplasmic shuttling transporter(s). Finally, a domain near
the C terminus recruits an interleukin-1-responsive kinase that
phosphorylates K protein in a nucleic acid-dependent
fashion (3, 4). Although K protein is already known to contain a number
of binding domains, it seems that there are potentially others that
remain to be identified. The abundant and ubiquitous expression of K
protein, its multimodular structure, its potential to oligomerize via
its two different dimerization domains, and its apparent involvement in
a wide range of processes responsible for transcription, translation,
and signal transduction suggest that K protein may act as a scaffold or
docking platform. Alternatively, K protein may be a multifunctional
factor that is involved in processes that are not directly related. In either scenario the function of K protein is likely to be regulated by
post-translational modification and by cognate nucleic acid motifs.
K protein is phosphorylated in vivo and in vitro
on serine and threonine residues (5, 23). At least in part, this
phosphorylation is mediated by an associated kinase(s) that can respond
to treatment of cells with interleukin-1 and other agents (4, 5). Thus, K protein phosphorylation is likely to play a key role in the regulation of its activity. This postulate is supported by the observation that the binding of K protein to poly(C) in
vitro is diminished by phosphorylation (23). Moreover, in
hepatocytes following systemic administration of lipopolysaccharide
into rats, there was a complete dissociation of the transcriptional
factor C/EBP The PKC family of enzymes transduce intracellular signals that regulate
many different intracellular processes (26). This heterogenous family
of enzymes is divided into three classes based on their
Ca2+ and lipid requirements (27). The conventional PKCs
( Cell Line--
The murine thymoma EL-4 6.1 C10 (31) and human
thymoma Jurkat cell line was grown in suspension. Cells were grown at
37 °C in complete RPMI 1640 medium supplemented with 5% fetal calf serum or Fetal Clone (Hyclone), 2 mM glutamine, 50 µM Reagents--
The bacterial expression vector pGEX-KT was
provided by Dr. J. Dixon (University of Michigan). Glutathione-agarose
beads, reduced glutathione, and PMA were obtained from Sigma. Fast Flow Protein A-Sepharose was obtained from Amersham Pharmacia Biotech. Polyclonal anti-K protein antibody 54 was made in rabbits as described previously (4). Monoclonal antiphosphotyrosine and polyclonal anti-PKC List of Buffers--
The following buffers were used: PKC
phosphorylation buffer, 50 mm Tris, pH 7.5, 10 mM
Extraction of Cytoplasmic and Nuclear Proteins--
Nuclear and
cytoplasmic extracts were prepared by a modified version of the method
of Dignam et al. (35) as described previously (5). In
addition to 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin, lysis,
extraction, and dilution buffers contained the following phosphatase
inhibitors (all from Sigma): 30 mM p-nitrophenyl
phosphate, 10 mM NaF, 0.1 mM
Na3VO4, 0.1 mM
Na2MoO4, and 10 mM
K Protein Plasmids Constructs--
GST-K, GST-K3, GST-K,
GST-K10, GST-K12, GST-K13, and GST-K14 were constructed as described
(3). For the GST-K31 deletion mutant, polymerase chain reaction was
used to introduce a BamHI site immediately upstream of
Met240 and to introduce a stop codon followed by an
EcoRI site downstream of Val337. GST-K31 was
created by cloning this excised fragment into pGEX-KT. For the
GST-K Synthesis and Purification of GST-K Constructs--
GST fusion
proteins were expressed in either BRL or BL21 (DE3) pLysS cells
(Novagen, Madison, WI) using a modified manufacturer's protocol.
Transformed cells were grown until they reached
A600 = 0.6 and then were treated for 3 h
with 1.0 mM isopropyl- In Vitro Transcription and Translation--
In vitro
transcription and translation was performed using the TNT T7 Quick
Coupled Transcription/Translation System as per the manufacturer's
protocol (Promega, Madison, WI)
Binding to Beads Bearing GST-K Proteins--
Binding to beads
was carried out by mixing glutathione beads with the standard binding
buffer containing a given protein or RNA at 4 °C. After extensive
washes, proteins were eluted from the beads by boiling in 1×
SDS-loading buffer (36) and were then loaded on a 10% SDS gel and autoradiographed.
Transient Transfections--
COS cells were grown in Dulbecco's
minimal essential medium supplemented with 10% fetal calf serum to
approximately 60-75% confluency in 100-mm diameter dishes and were
transfected using SuperFect Transfection Reagent as per the
manufacturer's protocol (Qiagen Inc., Santa Clarita, CA).
K Protein Is Phosphorylated in Vitro by PKC Mapping of K Protein Site That Is Phosphorylated in Vitro by
PKC--
A series of GST-K deletion mutants (Fig.
2A) (3) was used to identify a
domain that is phosphorylated by PKC
We used the above approach to determine whether K protein is a
substrate for other PKC isoforms. Beads bearing different GST-K protein
mutants (Fig. 3A) were
phosphorylated by a partially purified mixture of PKC PMA-inducible Phosphorylation of K Protein in Vivo--
K protein
is phosphorylated in vivo on serine residues (5). Thus, to
determine whether Ser302 can also be phosphorylated
in vivo, we compared the levels of phosphorylation of
wild-type Flag-K and of point mutant Flag-KS302A
(Ser302 K Protein Binds PKC Role of Ser302 on the in Vitro Interaction of K Protein
with PKC
Although under present conditions mutation of Ser302 had no
detectable effect on the in vitro interaction of K protein
with PKC Tyrosine Phosphorylation of K Protein Modulates Its Interaction
with PKC
The combination of
H2O2/Na3VO4 stimulates
tyrosine kinases in a myriad of cell types (38). To test the effects of
tyrosine phosphorylation of K protein on in vivo binding of
PKC
Next we tested whether PKC Poly(C) RNA Disrupts the Native K Protein·PKC K protein has previously been shown to be phosphorylated in
vivo, both constitutively and inducibly, by serine/threonine
kinases (5, 23). Until now, casein kinase II was the only kinase known
to phosphorylate K protein (24, 39). In the present study, we
demonstrate that K protein forms a complex with PKC We have identified Ser302 as a major site of
phosphorylation by PKC K protein binds PKC Based on what is already known about K protein and its molecular
partners, there are a number of specific scenarios that can be
envisaged where PKC PKC What is the significance of the observation that many factors that
interact with K protein are recruited by the KI domain? At least two
models can be construed. The two dimerization domains may allow K
protein to form higher order structures. If so, oligomerized K protein
would contain a number of KI domains that could engage the same or
different partners permitting uni- or multi-lateral cross-talk. It has
been suggested that K protein may be involved in the transport of
mRNA (15). Because K protein has the ability to shuttle between the
nucleus and cytoplasm (15), it may not only serve to transport RNA, but
it may also shuttle proteins. In that case, the KI domain may serve as
a docking site for transport of these factors between the nuclear and
cytoplasmic compartments. Whatever the role of the KI domain may be,
the activity of this region toward some of the partners is regulated by
growth factors, cytokines, and other extracellular stimuli that could
exert their effect through the activation of PKC In summary, we have shown that PKC and by other PKCs. Deletion analysis and site-directed
mutagenesis revealed that Ser302 is a major K protein site
phosphorylated by PKC
in vitro. This residue is located
in the middle of a short amino acid fragment that divides the two
clusters of SH3-binding domains. Mutation of Ser302
decreased the level of phosphorylation of exogenously expressed K
protein in phorbol 12-myristate 13-acetate-treated COS cells, suggesting that Ser302 is also a site for PKC-mediated
phosphorylation in vivo. In vitro, PKC
binds K protein
via the highly interactive KI domain, an interaction that
is blocked by poly(C) RNA. Mutation of Ser302 did not alter
the K protein-PKC
interaction in vitro, suggesting that
phosphorylation of this residue alone is not sufficient to alter this
interaction. Instead, binding of PKC
to K protein in
vitro and in vivo was greatly increased by K protein
phosphorylation on tyrosine residues. The ability of PKC
to bind and
phosphorylate K protein may serve not only to alter the activity of K
protein itself, but K protein may also bridge PKC
to other K protein molecular partners and thus facilitate molecular cross-talk. The regulated nature of the PKC
-K protein interaction may serve to meet
cellular needs at sites of active transcription, RNA processing and
translation in response to changing extracellular environment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mediated transcription of the agp gene
(11) and inhibits Sp-1-mediated activation of the neuronal nicotinic acetylcholine receptor promoter (12). As one of the constituents of the
hnRNP particle, K protein may be involved in the processing of
pre-mRNA (13, 14). K protein shuttles between the nucleus and
cytoplasm and therefore could serve as a vehicle that is involved in
RNA transport (15). K protein-mediated silencing of 15-lipoxygenase mRNA (16) represents an example of K protein involvement in the
regulation of translation. Involvement of K protein in translational processes is further supported by its association with the elongation factor 1
(1). The association of K protein with tyrosine kinases (2,
3) and with Vav (6, 7) may reflect involvement of K protein in signal
transduction. Alternatively, the Vav- and/or tyrosine kinases-K protein
interaction may regulate K protein transcriptional and/or translational
activity. Considering the diversity of K protein molecular
interactions, it is not surprising that new reports are emerging
implicating K protein involvement in viral processes. For example, K
protein has been shown to functionally interact with hepatitis C virus
core protein (17) and to regulate translation of the human
papillomavirus type 16 L2 mRNA (18).
from K protein (11), a process that may reflect
phosphorylation of K protein. Analysis of the K protein amino acid
sequence reveals a number of potential phosphorylation sites by casein
kinase II, protein kinase C (PKC), and
tyrosine kinases. Indeed, in vitro, K protein is an
excellent substrate for casein kinase II (24). However, casein kinase
II is typically a constitutively active enzyme (25), making its role in
the inducible phosphorylation of K protein in vivo less
likely than the role of inducible serine/threonine kinases such as the
PKC family of enzymes.
,
, and
) require phosphatidylserine, diacylglycerol, or
phorbol 12-myristate 13-acetate (PMA), and Ca2+; the novel
subgroup of PKCs (
,
,
, µ, and
) are
Ca2+-independent but require the other two co-factors; and
finally the least understood subgroup of PKCs (
and
) are both
Ca2+- and diacylglycerol-independent. Among the PKC
isoenzymes, PKC
has unique properties that suggest a functional
connection to K protein. First, PKC
phosphorylates elongation factor
1
(28), a factor that binds K protein (29). Second, K protein
interacts with Src-tyrosine kinases, a class of enzymes that
phosphorylate PKC
(30). Third, K protein contains several potential
PKC phosphorylation sites. Because K protein contains sites for PKC
phosphorylation and because PKC
and K protein share molecular
partners, in this study we explored the possibility that K protein is a
PKC
substrate.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 100 units/ml penicillin, 0.01%
streptomycin, and humidified with 7/93% C02/air gas
mixture. The monkey COS cells (32) were grown in Dulbecco's minimal
essential medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 0.01% streptomycin and humidified with 7/93% C02/air gas mixture.
antibody was purchased from Santa Cruz and anti-PKC
monoclonal antibody was purchased from Transduction Laboratories. Baculovirus expressed and spleen PKC
were produced as described previously (33, 34).
-mercaptoethanol, 1 mM PMA, 10 mg phosphatidylserine,
18.75 mM ATP, and 3.7 mCi of [
-32P]ATP;
PKC binding buffer, 50 mM Tris, pH 7.5, 10 mM
-mercaptoethanol, and 1 mM PMA; Lck phosphorylation
buffer, 20 mM Tris-HCl, pH 7.5, 10 mM
MnCl2, 100 µM ATP, 1 mM
dithiothreitol, and 0.1% Triton X-100; Escherichia coli
extraction buffer, 50 mM Tris-HCl, pH 8.0, 2 mM
EDTA, 0.5 mM dithiothreitol, 10 mg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride; standard binding buffer, 5 mM Tris, pH 7.5, 1 mM EDTA, 25 mM
NaCl, and 0.05% Nonidet P-40; HKMT buffer, 10 mM Hepes, pH
7.5, 2 mM MgCl2, 0.1%Triton X-100, and 100 mM KCl; washing buffer, 5 mM Tris-HCl, pH 7.5, 175 mM NaCl, 1.0 mM EDTA, and 1.0% Nonidet
P-40; SDS-loading buffer, 60 mM Tris-HCl, pH 6.8, 2% SDS,
10% glycerol, and 5%
-mercaptoethanol; TBST buffer, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05%
Tween 20; and IP buffer, 150 mM NaCl, 50 mM
Tris-HCl, pH 7.5, 5 mM EDTA, 0.5% Nonidet P-40, and 1.0%
Triton X-100.
-glycerophosphate. Protein content was measured using the DC method (Pierce).
PB deletion mutant, pGEX-K was cut with PpuMI and BglII, then filled in with Klenow fragment, and religated to
create the GST-K
PB construct. For Flag-K for mammalian expression, K protein was excised from pM1-K (22) with EcoRI and
SalI and then ligated into p18Flag that had been cut with
EcoRI and XhoI, creating the recombinant Flag-K.
The point mutations of Ser302 to Ala or Glu in GST-K,
GST-K31, and Flag-K were generated using the QuickChange Site-Directed
Mutagenesis kit (Stratagene). All plasmids were purified by CsCl
gradient before use in transient transfections, and the mutations were
confirmed by automated sequencing using the DyeDeoxy Terminator Cycle
Sequencing kit (PE Applied Biosystems, Foster City, CA).
-D-thiogalactoside. Following freezing and thawing, pellets were resuspended in E. coli extraction buffer and sonicated. Fusion proteins were
recovered in the supernatant after centrifugation at 14,000 rpm for 30 min (4 °C). GST-K and GST-K deletion mutants were then purified on a
glutathione column as described previously (3).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
--
K protein is
phosphorylated in vivo and in vitro on serine
residues by a kinase(s) with which it forms a complex (5, 23). Analysis
of K protein amino acid sequence reveals a number of potential sites
for phosphorylation by PKC. To test whether K protein is a substrate
for this class of enzymes, purified bacterially expressed full-length
GST-K fusion protein was phosphorylated in solution by purified porcine
spleen PKC
in the presence of [
-32P]ATP. The kinase
reaction was terminated by boiling the sample with loading buffer and
then separated by SDS-PAGE. Phosphorylation of GST-K protein was
assessed by scintillation counting of the 32P-labeled GST-K
bands cut from the gel (Fig. 1). The
results showed that PKC
phosphorylates K protein in vitro
and that the rate of phosphate incorporation was dependent on GST-K
protein concentration. Under these conditions, phosphorylation of GST-K
had a Vmax of 286 pmol phosphate/min/mg PKC
and a Km of 1.9 nM (Fig. 1B).
These results show that under these conditions the velocity of
phosphorylation of GST-K by PKC
was relatively slow, but the low
Km suggests that K protein may bind PKC
with a
high affinity. As expected, phosphorylation of K protein by PKC
is dependent on phosphatidylserine and PMA and does not require
Ca2+ (data not shown).
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Fig. 1.
Phosphorylation of GST-K protein by
PKC . Different amounts of purified GST-K
protein were phosphorylated in solution (10 min at 30 °C) with 3.5 milliunits of PKC
(45.5 ng of protein, purified from porcine spleen)
in PKC phosphorylation buffer. (1 unit of kinase activity equals 1 nmol
of phosphate/min incorporated into histone IIIS). The reaction mixture
was separated by SDS-PAGE, GST-K protein bands were cut out, and
radioactivity was measured. Rates of phosphate incorporation were
plotted as a function of GST-K protein concentration (A),
and 1/V was plotted as a function of 1/[GST-K] in B. The
y axis intercept of the double-reciprocal plot
(B) is 1/Vmax = 77 pmol
min
1 or Vmax = 0.013 pmol/min/45.5
ng PKC
286 pmol/min/mg PKC
. The x axis
intercept of the double-reciprocal plot is 1/Km =
0.53 nM
1 or Km = 1.9 nM.
. Glutathione beads bearing
either full-length GST-K or one of the deletion mutants were
phosphorylated by baculovirus expressed PKC
(28, 37). The kinase
reaction was terminated by washing the beads with HKMT buffer, proteins
were eluted from the beads by boiling in SDS loading buffer, and the
level of phosphorylation of GST-K fusion proteins was assessed by
SDS-PAGE and autoradiography (Fig. 2B). In addition to
phosphorylating the full-length GST-K, PKC
also phosphorylated
GST-K13 (a.a. 1-337), GST-K3 (a.a. 171-337), and GST-K31 (a.a.
240-337) deletion mutants. In contrast, the deletion mutants GST-K12
(a.a. 1-209) and GST-K7 (a.a. 318-464) were not phosphorylated at
all. These results indicated that the PKC
site(s) is contained
within the GST-K31 deletion mutant (a.a. 240-337). This domain
contains three serines, Ser276, Ser284, and
Ser302, and no threonines. Ser302 is located
within a good consensus for PKC-mediated phosphorylation, Arg-Gly-Gly-Ser-Arg-Ala-Arg (24, 28). To test whether
Ser302 is the site that is phosphorylated in
vitro by PKC
, site-directed mutagenesis was used to mutate this
residue to either Ala (GST-KS302A and
GST-K31S302A) or Glu (GST-KS302E and
GST-K31S302E) (Fig. 2A). These mutants were
tested as substrates for phosphorylation by PKC
. As before, the
phosphorylation reactions were carried out on beads bearing GST-K
fusion proteins (Fig. 2). Autoradiographs from these phosphorylation
reactions are shown in Fig. 2C. In sharp contrast to the
full-length GST-K, the point mutants GST-KS302A and
GST-KS302E were poorly or not at all phosphorylated by
PKC
. Although the GST-K31S302A and
GST-K31S302E mutants were phosphorylated, the level of
phosphorylation of these Ser302 mutants was much lower than
that observed with the wild-type GST-K31. These results indicate that
Ser302 is the major site for phosphorylation of GST-K
protein by PKC
in vitro, whereas Ser277
and/or Ser284 are only minor site(s). This is supported by
the observation that an internal deletion mutant that lacks the
proline-rich SH3-binding domains, GST-K
PB (a.a. 288-321 deleted),
was a very poor substrate for PKC
(data not shown).
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Fig. 2.
Mapping of the K protein site that is
phosphorylated in vitro by
PKC . 20 µl of glutathione beads bearing
either a full-length GST-K or one of the K protein mutants fused to GST
(A) were suspended in phosphorylation buffer containing 0.5 µl of baculovirus expressed PKC
, and the phosphorylation reaction
was carried out as in Fig. 1. Afterward, the beads were washed once
with 200 µl of standard binding buffer. 32P-Labeled
proteins were eluted from the beads by boiling in SDS-loading buffer
and then separated by SDS-PAGE. The gels were stained
(Coomassie) and autoradiographed (32P)
(B). Molecular mass markers are shown in kDa.
,
, and
.
As with PKC
, GST-K and GST-K31 were phosphorylated by PKC
,
,
and
, whereas GST-K7 (a.a. 318-464) was not phosphorylated at all,
and the level of phosphorylation of the internal deletion mutant that
lacks the two clusters of the proline-rich SH3-binding domains (3),
GST-K
PB (a.a. 288-321 deleted) was very low (Fig. 3B).
Because the deleted a.a. 288-321 region contains no other
phosphorylation sites, the markedly decreased level of phosphorylation
of the GST-K
PB mutant suggests that Ser302 is also a
major K protein site for phosphorylation by the
calcium-dependent PKC
,
, and
. The level of
PKC
-,
-, and
mediated phosphorylation of
GST-KS302A, GST-KS302E,
GST-K31S302A, and GST-K31S302E mutants was
lower compared with their respective wild-type GST-K fusion proteins
(Fig. 3C), providing further evidence that
Ser302 is a major site of phosphorylation by these PKC
isoforms in vitro.
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Fig. 3.
In vitro phosphorylation of K
protein Ser302 by PKC ,
, and
. 20 µl of
glutathione beads bearing either full-length K protein or one of the K
protein mutants fused to GST (A) were phosphorylated by
PKC
,
, and
purified from bovine brain (46) under conditions
described for PKC
. Phosphorylated proteins were analyzed by SDS-PAGE
and autoradiography (B and C) as in Fig.
2.
Ala) that were co-expressed with HA-PKC
in
COS cells. Transfected cells were metabolically labeled with
[32P]orthophosphate and were then treated with or without
10
7 M PMA for 1 h. Following treatment,
cells were harvested, and cytoplasmic and nuclear extracts were
prepared as described previously (5). Equal amounts of extracts were
precipitated with either pre-immune or immune anti-K protein serum
(antibody 54). The immunoprecipitates were separated by SDS-PAGE and
were then electrotransferred to Immobilon-P membrane (Millipore,
Bedford, MA) and were analyzed by autoradiography (Fig.
4, 32P) and by Western
blotting using anti-K protein serum (Fig. 4,
K). The
autoradiograph revealed that in the cytoplasm the constitutive level of
phosphorylation of Flag-K was higher that of Flag-KS302A.
In the nuclear fraction, phosphorylation of Flag-K was PMA-inducible, whereas phosphorylation of Flag-KS302A was not. In both the
nucleus and the cytoplasm, the level of Flag-K PMA-inducible
phosphorylation was higher than the level of Flag-KS302A
phosphorylation in PMA-treated cells. Unlike the exogenously expressed
Flag-K proteins, the levels of PMA-inducible phosphorylation of
endogenous K protein (Fig. 4, lower band in the
autoradiograph marked K), in the Flag-K and
Flag-KS302A transfected cells were similar. These
experiments provide evidence that Ser302 can be
phosphorylated in vivo, a reaction that may, in part, be
mediated by PKC
and/or other PKC isoenzymes.
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Fig. 4.
In vivo 32P labeling
of wild-type and Ser302 Ala mutated Flag-K
protein. A, COS cells were transiently co-transfected
(2 µg of each plasmid/100 mm plate) with a Flag tag mammalian
expression plasmid containing either wild-type K protein, Flag-K, or K
protein with Ser302
Ala mutation,
Flag-KS302A, along with expression vector (pCEV27)
containing HA-PKC
. After an overnight transfection, cells were
washed three times with phosphate free RPMI media, and then incubated
with 0.3-0.5 mCi of [32P]orthophosphate/plate. After
24 h, cells were treated with or without 10
7
M PMA for 60 min. Cells were then harvested, and extracts
were prepared. Nuclear and cytoplasmic extracts diluted to 200 µl
with HKMT-PI were mixed for 1 h (4 °C) with 10 µl of protein
A-agarose beads bearing either pre-immune (lanes 1 and
6) or immune anti-K protein polyclonal rabbit antibody 54 (lanes 2-5 and 7-10). After IP, beads were
washed for 5 min with 0.2 ml of HKMT-PI buffer, followed by 5 min with
0.2 ml of wash buffer. Proteins were eluted by boiling in SDS-loading
buffer, separated by SDS-PAGE, and then electrotransferred onto
Immobilon-P membrane. Membranes were immunostained with anti-K protein
antibody 54 (1:10,000 dilution) (lower panel,
K), and were then autoradiographed (upper
panel, 32P). The exogenous Flag-K and
Flag-KS302A and the endogenous K protein are indicated.
B, COS cells were transfected with either Flag or Flag-K,
extracts were prepared and K and Flag-K proteins were
immunoprecipitated with anti-K antibody as above. Immunoprecipitated
proteins were resolved by SDS-PAGE, and Western blotting was done with
either anti-K protein (lanes 1 and 2) or
anti-Flag (lanes 3 and 4) antibodies. The
endogenous K protein (K) and Flag-K fusion protein
(Flag-K) are marked.
through Its Highly Interactive
Domain--
The low Km for the phosphorylation of
GST-K by PKC
(Fig. 1) suggests that the two proteins may bind one
another with a high enough affinity to form detectable complexes. To
test such a possibility, beads bearing GST or GST-K fusion proteins (Fig. 5) were mixed with baculovirus
expressed PKC
, and after binding and washing, bound proteins were
eluted from the beads by boiling in loading buffer. Proteins eluted
from the beads were separated by SDS-PAGE and electrotransferred to
Immobilon-P membrane for immunostaining with anti-PKC
antibody. To
ensure that beads contained similar levels of GST fusion proteins,
another gel was stained with Coomassie. These results showed that beads
bearing GST-K protein, but not beads bearing GST alone, pulled down
PKC
, indicating that K protein binds PKC
in vitro.
Several GST-K deletion mutants were used to map the K protein domain
that binds PKC
in vitro. GST-K13 (a.a. 1-337) and
GST-K31 (a.a. 240-337) bound PKC
as effectively as the full-length
GST-K protein, whereas deletion mutants GST-K7 (a.a. 318-464), GST-K10
(a.a. 318-382), GST-K12 (a.a. 1-209), and GST-K14 (a.a. 406-464) did
not bind PKC
at all. The deletion mutant that lacks the two clusters
of SH3-binding domains, GST-K
PB (deleted a.a. 288-321 fragment), bound PCK
weakly. These results indicate that the domain of K protein that binds PKC
in vitro is contained within the
a.a. 240-337 region and that the proline-rich region may be important in this interaction. The a.a. 240-337 K protein domain interacts with
a number of known K protein partners. In addition to recruiting PKC
,
this domain also mediates the binding of the transcriptional repressors
Zik1 (21) and Eed (22), several tyrosine kinases (3), and the
proto-oncoprotein Vav (6). It is likely that this domain recruits many
other factors that are involved in signal transduction and gene
expression. We designate this region as the KI domain, for
K protein interactive domain.
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Fig. 5.
Binding of PKC to K
protein in vitro. 20 µl of glutathione beads, suspended in
PKC binding buffer and bearing either full-length or one of the K
protein mutants fused to GST (A) were mixed with 0.2 µl of
baculovirus expressed PKC
for 1 h at 4 °C. After the binding
reaction, the beads were washed twice with 1.0 ml of PKC binding buffer
and once with washing buffer. Beads were boiled in SDS-loading buffer,
the sample was divided into two equal aliquots, and the eluted proteins
were separated by SDS-PAGE. One of the gels was stained with Coomassie
(B), while proteins from the other gel were
electrotransferred to Immobilon-P membrane. The blot was immunostained
with polyclonal anti-PKC
antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) (B, PKC
). Molecular mass markers are shown in
kDa.
in the Presence or Absence of RNA--
Ser302
is located within the highly interactive KI domain, in the middle of a
short amino acid stretch that splits the two clusters of SH3-binding
domains. The location of Ser302 suggests that this residue
might play a role in the regulation of K protein interaction with
PKC
and/or other partners. To test this possibility, beads bearing
either wild-type GST-K protein or GST-K protein with mutated
Ser302, GST-KS302A or GST-KS302E,
were mixed with baculovirus expressed PKC
, and the binding was
assessed as before by SDS-PAGE and Western blotting with an anti-PKC
antibody. Results illustrated in Fig.
6A show that mutating Ser302 to either Ala or Glu did not alter the ability of K
protein to recruit PKC
. The in vitro interaction of K
protein with many of its molecular partners can be regulated by cognate
RNA, such as poly(C) RNA, or a cognate DNA, such as the
B, motif (4, 21). Thus, we also tested whether mutation of Ser302 alters
the affinity of K protein-PKC
complex when K protein is bound to
poly(C). These results showed that poly(C) RNA abrogated the in
vitro interaction of K protein with PKC
independently of
Ser302 and suggests that the ability of K protein to bind
poly(C) is not altered by the mutation of Ser302.
Abrogation of the in vitro association between K protein and PKC
by poly(C) is similar to the observations of other K protein partners that are recruited to K protein by the KI domain (21).
View larger version (36K):
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Fig. 6.
Analysis of the effects of Ser302
mutation on the binding of PKC
(A) and Eed (B) in the presence
or absence of RNA. 20 µl of glutathione beads bearing either
wild-type full-length GST-K (GST-K) or GST-K with either
Ser302
Ala (GST-KS302A) or
Ser302
Glu (GST-KS302E) mutations were
preincubated for 30 min (4 °C) in 100 µl of standard binding
buffer containing either no RNA (lanes 1, 4, and
7) or 10 µg/ml of either poly(A) (lanes 2,
5, and 8) or poly(C) (lanes 3,
6, and 9). Beads were washed twice with 1.0 ml of
washing buffer and once with 1.0 ml of PKC binding buffer.
A, washed beads were resuspended in 100 µl of PKC binding
buffer, and the bead suspension was mixed with 0.2 µl of baculovirus
expressed PKC
for 1 h (4 °C). Beads were washed twice with 1 ml of PKC binding buffer and once with 1 ml of washing buffer. Proteins
were eluted by boiling in SDS-loading buffer. The eluted proteins were
separated by SDS-PAGE in duplicate gels. One gel was electrotransferred
onto Immobilon-P membrane and was blotted with a polyclonal anti-PKC
antibody (PKC
), whereas the other was stained with Coomassie
(GST-K). 50% of PKC
load was run in lane 10.
B, washed beads, loaded with or without RNA, were
resuspended in 100 µl of HKMT buffer. The bead suspension was mixed
with 0.2 µl of in vitro synthesized 35S-Eed
for 1 h (4 °C). Beads were washed twice with 1 ml of HKMT
buffer and once with 1 ml of washing buffer. Proteins were eluted by
boiling the beads in SDS-loading buffer, and the eluates were separated
by SDS-PAGE. Gels were stained with Coomassie (GST-K,
lower panel) and were autoradiographed
(35S-Eed, upper panel). 50% of the
amount of 35S-Eed used in pull down assays was run in
lane 10 (load).
or on its ability to bind poly(C), this residue may play a
role in the engagement of other K protein partners. For example, the protein Eed is recruited to K protein by the KI domain (22), an
interaction that could be modulated by Ser302.
35S-Eed was mixed with GST-K, GST-KS302A, or
GST-KS302E beads that had been pre-equilibrated with or
without RNA. The K protein-bound 35S-Eed was analyzed by
SDS-PAGE and autoradiography (Fig. 6B). These results
revealed that mutation of Ser302 to Ala diminished the
ability of 35S-Eed to be recruited by K protein, and the
Ser302 to Glu302 mutation had even more of a
blocking effect on this association. These results suggest that the
recruitment of some of the K protein partners may be regulated by
phosphorylation of Ser302.
in Vitro and in Vivo--
Phosphorylation of K protein by
the tyrosine kinase Lck regulates K protein interaction with a number
of its molecular partners.2
We tested whether tyrosine phosphorylation also regulates the association of PKC
with K protein. Glutathione beads bearing GST-K
protein were incubated in Lck phosphorylation buffer with or without
baculovirus Lck. After extensive washes, the beads were incubated with
35S-PKC
synthesized in the cell-free system (Promega).
The beads were washed again, and the bound proteins were eluted by
boiling, separated by SDS-PAGE, and transferred to Immobilon-P
membrane. Fig. 7A illustrates
results from this experiment. Lck-mediated tyrosine phosphorylation of
K protein (upper blot) greatly enhanced the in
vitro binding of 35S-PKC
to K protein (Fig.
7A, lanes 1 and 2, middle blot). The gels also showed that tyrosine phosphorylation of K protein shifted its
electrophoretic mobility and the protein band became wider (Fig.
7A, compare lanes 1 and 2,
bottom blot). The marked changes in the electrophoretic
mobility indicate a significant change in K protein structure that
coincides with strong recruitment of PKC
to K protein. Not
unexpectedly, the Lck-mediated phosphorylation of K protein has
different effects on the interaction of K protein with different
molecular partners. Although the K protein binding of PKC
(Fig.
7A), as well as the binding of tyrosine kinases and Vav, is
increased by tyrosine phosphorylation of K protein, the K protein
binding of Zik-1 and Eed is blocked by tyrosine phosphorylation.
Moreover, the K protein binding of elongation factor 1
and
TATA-binding protein is not affected by K protein tyrosine
phosphorylation.2
View larger version (16K):
[in a new window]
Fig. 7.
Tyrosine phosphorylation of K protein
stimulates its association with PKC .
A, 15 µl of glutathione beads bearing GST-K protein were
incubated in 50 µl of Lck phosphorylation buffer for 2 h at
30 °C without (lane 1) or with (lane 2) 2 µl
of recombinant baculovirus Lck. The phosphorylation reaction was
terminated by washing the beads four times with 1 ml of HKMT buffer.
0.6 ml of HKMT buffer containing 6 µl of 35S-PKC
synthesized in cell-free system was centrifuged at 12,000 × g for 5 min at 4 °C. 200 µl of the spun solution was
added to each aliquot of the washed beads, and the bead suspension was
mixed for 30 min at 4 °C. The beads were then washed four times with
HKMT buffer, and the bound proteins were eluted by boiling in loading
buffer. Eluted proteins were separated by SDS-PAGE and transferred to
polyvinylidene difluoride membrane. After staining with amido black
(lower insert), 35S-labeled proteins were
visualized by autoradiography (middle insert). The membrane
was immunostained (IS, upper insert) with
monoclonal anti-phosphotyrosine antibody (PY99, 1:200 dilution, Santa
Cruz Biotechnology) and alkaline phosphatase-conjugated goat anti-mouse
antibody (1:1000 dilution, Santa Cruz Biotechnology). B:
Jurkat cells (400 × 106 at 1.0 × 106 cells/ml) were treated with 3.5 mM
H2O2/0.1 mM
Na3VO4. At the given time points, cells were
lysed with IP buffer for 30 min on ice. Lysates were centrifuged for 30 min at 13,000 rpm at 4 °C. 100 µl of cell lysate was sonicated for
60 min (4 °C) with 3 µl of either preimmune (lanes
1-3) or immune (lanes 4-6) anti-K protein antibody 54 (
K) rabbit serum. After sonication, the samples were
centrifuged for 5 min at 13,000 × g (4 °C), and the
supernatants were added to 20 µl of protein A/G beads (Santa Cruz
Biotechnology). The suspensions were mixed for 30 min (4 °C), then
the beads were washed four times with 1 ml of IP buffer, and the
proteins were eluted by boiling in 50 µl of loading buffer and
resolved by SDS-PAGE. After electrotransfer to Immobilon-P membrane,
immunostaining was done either with (lower insert) anti-K
protein serum 54 (1:5000 dilution), alkaline phosphatase-conjugated
anti-rabbit antibody (1:3000 dilution, Bio-Rad), and
5-bromo-4-chloro-indolyl-phosphatase/nitroblue tetrazolium phosphatase
substrate (Kirkegaard & Perry Laboratories) or with (upper
insert) anti-phosphotyrosine monoclonal (PY99) as in A
(middle blot). C, 200 µl of lysates from given
time points of
H2O2/Na3VO4-treated
Jurkat cells were sonicated for 2 h (4 °C) with 50 µl of
either anti-PKC
monoclonal antibody (
PKC
,
lanes 1-3) (Transduction Laboratories) or 50 µl of
anti-Flag monoclonal antibody (
Flag, lanes
4-6) (Santa Cruz). After sonication, 20 µl of protein A/G beads
were added to each sample. The suspensions were mixed for 2 h
(4 °C), the beads were washed four times with 1 ml of IP buffer, and
the proteins were eluted by boiling in loading buffer. Eluted proteins
were resolved on SDS-PAGE, and after electrotransfer to Immobilon-P
membrane, immunostaining of K protein was carried out with anti-K
protein antibody 54 (
K) using the sandwich technique
(upper insert). To assess the levels of PKC
in the
immunoprecipitates (middle insert), Immobilon-P membranes
were immunostained with a monoclonal anti-PKC
antibody (1:200
dilution) (Transduction Laboratories), alkaline phosphatase-conjugated
goat anti-mouse antibody (1:1000 dilution) (Santa Cruz), and
5-bromo-4-chloro-indolyl-phosphatase/nitroblue tetrazolium. To assess
tyrosine phosphorylation of PKC
(lower insert) membranes
were immunostained with anti-phosphotyrosine monoclonal antibody (1:200
dilution) (PY99) and horseradish peroxidase-conjugated goat anti-mouse
secondary antibody (1:2000 dilution) (Amersham Pharmacia Biotech) and
developed with ECL (Amersham Pharmacia Biotech).
, Jurkat cells were treated with
H2O2/Na3VO4. At given
time points shown in Fig. 7, cell lysates were prepared, and
immunoprecipitations were carried out with either pre-immune or immune
anti-K protein serum. After SDS-PAGE and electrotransfer, Immobilon-P
membranes were immunostained with either anti-phosphotyrosine (Fig.
7B, lower blot) or anti-K protein (Fig. 7B, upper
blot). Anti-phosphotyrosine immunostaining showed that treatment
of Jurkat cells with the combination of these agents induced a
transient increase in tyrosine phosphorylation of K protein; there was
a low constitutive level of K protein tyrosine phosphorylation, an
easily detectable increase after 15 min of treatment, and a decrease
after 60 min (Fig. 7B, upper blot). As in
vitro, the electrophoretic mobility of in vivo tyrosine
phosphorylated K protein was slower (Fig. 7B, compare lanes 4 and 5, upper blot).
exists in a complex with K protein
in vivo and, if so, whether this association is regulated by tyrosine phosphorylation. Immunoprecipitations were carried on cell
lysates from Jurkat cells treated with
H2O2/Na3VO4 using a
monoclonal anti-PKC
antibody. A monoclonal anti-Flag antibody was
used as a control. Fig. 7C (upper blot) shows
that K protein co-immunoprecipitated with PKC
from cell lysates; a
very low level of K protein co-immunoprecipitated with PKC
from cell
lysates of untreated cells (lane 1); there was a large
increase in co-immunoprecipitated K protein after 15 min of treatment
(lane 2) and a significant decrease at 60 min (lane
3). These results suggest that in vivo there is a low
constitutive level of PKC
binding to K protein, an association that
is greatly enhanced by treatment of cells with
H2O2/Na3VO4. To assess
the levels of immunoprecipitated PKC
and to determine whether there
is tyrosine phosphorylation of PKC
, anti-PKC
immunoprecipitates
from Jurkat cell lysates were analyzed by SDS-PAGE and anti-PKC
(Fig. 7C, middle blot) and anti-phosphotyrosine
(Fig. 7C, bottom blot) immunostaining. These results showed that the amount of PKC
immunoprecipitated from cell
lysates progressively decreased after the treatment and that the
electrophoretic mobility of PKC
was slower (compare lanes 1-3). The amount and the electrophoretic mobility of the
immunoprecipitated PKC
reflect accurately the levels of PKC
found
in these cell lysates by Western blotting (data not shown). Although
the decrease in PKC
levels in cell lysates may reflect translocation
of this enzyme to the particulate fraction and/or proteolytic cleavage, the slower electrophoretic mobility may result from tyrosine
phosphorylation of PKC
. Because the in vitro binding of
PKC
to K protein is increased by tyrosine phosphorylation of K
protein (Fig. 7A) and because treatment of Jurkat cells with
H2O2/Na3VO4 stimulates tyrosine phosphorylation of K protein (Fig. 7B), the
enhanced association of PKC
with K protein in vivo is
likely the result, at least in part, of tyrosine phosphorylation of K
protein. Although in vitro the increased binding of PKC
to tyrosine phosphorylated K protein does not require tyrosine
phosphorylation of PKC
(Fig. 7A), treatment of cells with
H2O2/Na3VO4 stimulates
tyrosine phosphorylation of this enzyme (Fig. 7C,
lower blot), a modification that may contribute to the
enhanced PKC
-K protein association. Regardless of the specific
mechanisms responsible for the enhanced association, these results
illustrate that the binding of PKC
to K protein is regulated in
response to changes in the extracellular environment.
Complex--
The above results demonstrate that the in
vitro complex formation between recombinant K protein and PKC
is blocked by poly(C) RNA (Fig. 6), which tenaciously binds K protein.
Next we tested whether cognate RNA can disrupt the native PKC
-K
protein complex. Proteins from cytoplasmic extracts were precipitated
with either pre-immune or immune anti-K protein serum and protein A/G
beads. After a round of washing, proteins were eluted from the beads with either PKC buffer alone or PKC buffer containing either poly(A) or
poly(C) RNA. Eluates were then analyzed by SDS-PAGE followed by Western
blotting with a monoclonal anti-PKC
antibody. Immunostaining revealed that PKC
was eluted from beads bearing anti-K serum but not
from the pre-immune beads and that the amount of eluted PKC
was the
highest with poly(C) RNA (Fig. 8). This
finding suggests that in vivo the cognate nucleic acids have
the potential to regulate the PKC
-K protein association. Along with
the observation that tyrosine phosphorylation modulates the association
between K protein and PKC
(Fig. 7), the effect of poly(C) RNA on
this interaction (Figs. 6 and 8) provides further evidence that the
association between the two proteins is regulated.
View larger version (19K):
[in a new window]
Fig. 8.
Poly(C) RNA disrupts native K
protein-PKC complex. 2.0 mg of EL-4
cytoplasmic extracts containing 1% Triton X-100 were pre-cleared by
centrifugation for 30 min (4 °C) at 15,000 × g.
Supernatants were sonicated for 1 h (4 °C) with either 10 µl
of pre-immune or anti-K protein antibody 54 rabbit serum. The samples
were centrifuged again, and the supernatants were mixed with 40 µl of
bovine serum albumin-blocked protein A beads for 30 min (4 °C).
After precipitation, beads were washed three times with 1.0 ml of HKMT
buffer and were then mixed for 30 min (4 °C) with 50 µl of PKC
binding buffer containing 2 µg of bovine serum albumin without RNA
(lanes 1 and 4) or with 1 µg of either poly(A)
(lanes 2 and 5) or poly(C) (lanes 3 and 6). After elution, the suspension was centrifuged, and
the eluates were boiled in SDS-loading buffer. Eluted proteins were
separated by SDS-PAGE, electrotransferred to Immobilon-P membrane, and
then immunostained with anti-PKC
monoclonal antibody (50 µg/10 ml
TBST) (Transduction Laboratory). 20% of the total amount of PKC
directly immunoprecipitated from 2.0 mg of EL-4 cytoplasmic extracts
with anti-PKC
monoclonal antibody was run in lane
7.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(Figs. 6-8) and
can serve as its substrate (Figs. 1 and 2). K protein is not a specific
PKC
substrate because it can also be phosphorylated by PKC
,
,
and
(Fig. 3). In addition to the conventional (
,
, and
)
and novel PKCs (
,
,
, and µ), K protein may also be a
substrate for the atypical PKCs (
and
). In that regard, it is
notable that hnRNP A1 is a substrate for, and binds to, PKC
(40).
(Fig. 2) and by PKC (
,
, and
) (Fig.
3) in vitro. This site is also phosphorylated in
vivo in response to treatment of cells with PMA (Fig. 4),
suggesting that the in vivo phosphorylation of K protein is,
at least in part, mediated by PKC
and/or other PKCs.
Ser302 is located exactly in the middle of a stretch that
separates the two proline-rich domains that exhibit SH3 binding
activity (3). This strategic location suggests that Ser302
plays a role in the regulation of K protein interaction with some of
its partners. In support of this hypothesis is the observation that
mutation of Ser302 to Glu diminished the affinity of K
protein-Eed interaction in vitro (Fig. 6). Although the
mutation of Ser302 had no effect on K protein binding to
PKC
or RNA in vitro (Fig. 6), there may be other partners
in addition to Eed whose association with K protein are regulated by
this residue. It is also likely that phosphorylation of other sites in
conjunction with Ser302 may play a role in modulating the
binding of PKC
to K protein. Moreover, phosphorylation of
Ser302 may regulate cross-talk among factors that are
simultaneously engaged by K protein. For example, Vav binds K protein
and is tyrosine phosphorylated in response to cytokines (41).
Phosphorylation of Vav may occur in the context of K protein
simultaneously engaging Vav and a tyrosine kinase via the two clusters
of SH3-binding domains (3). If so, it is conceivable that the
juxtaposition of Vav and its tyrosine kinases may be altered by
PKC
-mediated phosphorylation of K protein Ser302. Such a
model could be applied to many of the K protein molecular partners.
with high enough affinity (Figs. 1 and 6) to
allow the two proteins to exist as detectable complexes in
vivo (Figs. 7 and 8). What is the physiological relevance of the
inducible nature of this association? First, the inducible binding is
likely to ensure effective phosphorylation of K protein by PKC
,
especially in response to a changing extracellular environment. Second,
the binding of PKC
to K protein may link this enzyme to its targets
or effectors that are concurrently present in the K protein
microenvironment. Third, because K protein shuttles between the nucleus
and cytoplasm (15), PKC
could be co-transported to specific
subcellular compartments by K protein. These three scenarios are not
mutually exclusive, and there may be other physiological meanings of
the PKC
-K protein association. Whatever the physiological role(s)
may be, the PKC
-K protein binding could be modulated by changes in
the extracellular environment that may include oxidative stress (Fig.
7, B and C) and acute phase reactions. The
in vivo interaction between the two partners may be further
regulated by cognate nucleic acids, such as specific RNA sequences.
These findings, in conjunction with the previous observations that K protein phosphorylation is modulated by interleukin-1 (5), and the
report that in intact organs K protein association with some of its
molecular partners is modulated by an acute phase reaction (11),
provide evidence that K protein function is regulated and responds to
the needs of the cell in the face of a changing external environment.
-K protein interaction may play a role in
determining cellular response. For example, in response to cytokines,
oxidative stress, or acute phase reaction, the Src family of tyrosine
kinases are activated, phosphorylating and then binding to K protein.
Tyrosine phosphorylation of K protein would then induce enhanced
binding of PKC
to K protein (Fig. 7). The simultaneous binding of a
tyrosine kinase and PKC
in the context of K protein would provide an
opportunity for cross-talk between these enzymes.
Tyrosine-phosphorylated and activated PKC
could then dissociate from
K protein and target other factors. Alternatively, K protein may
provide a platform that facilitates the ability of PKC
to target
those substrates that are simultaneously recruited by K protein. In
regard to these models, a number of PKCs, including PKC
(Fig.
7C), are tyrosine phosphorylated, a modification that
renders them phospholipid- and Ca2+-independent (42).
Because the recruitment of PKC
to K protein can be dramatically
altered in vitro by cognate RNA (Figs. 6 and 8), the K
protein-facilitated cross-talk between PKC
and its targets and/or
its effectors may be regulated by RNA in vivo. Moreover, K
protein serving as a nucleic acid-interacting docking platform would
facilitate molecular cross-talk at sites of active transcription,
translation, and other processes involving RNA and DNA. The recruitment
of PKC
to K protein is likely to be just one example of a more
general phenomenon involving the association of K protein with several
members of the PKC family of enzymes. This is supported by the
observations that PKC
,
, and
phosphorylate K protein (Fig. 3)
and by a report that K protein binds PKC
in vitro (43).
Moreover, because the yeast homologs of mammalian K protein and PKC
have been shown to be functionally linked (44), the K protein-PKC
interactions appear to be evolutionarily conserved in species as
diverse as yeast and mammals.
binds to a site within the highly interactive K protein region
hereby designated as the KI domain (Fig. 5). Besides PKC
, the KI
domain binds the Src family of kinases (3), the proto-oncoprotein Vav
(7), the transcriptional repressor Zik1 (21), the Polycomb group
protein Eed (22), and likely many other factors. Within the KI domain
these K protein partners may bind to the same or different sites.
However, not all K protein partners bind to the KI domain. For example,
TATA-binding protein binds K protein very strongly (8) through a region
that is different from the KI domain.3 Other examples of KI
domain-independent interaction includes the binding of RNA and DNA to
the KH domains (19, 45). Moreover, although the transcriptional factor
C/EBP
binds to a domain contained in the N-terminal half of the
molecule (11), an interleukin-1-responsive kinase binds to a domain in
the vicinity of the C terminus (3).
, Src-tyrosine
kinases, and other enzymes that phosphorylate sites located within the
KI domain.
binds and phosphorylates K
protein. These observations broaden the range of K protein interactions. PKC
targets Ser302, which is located in
the middle of what appears to be a highly interactive KI domain. The
ability of PKC
to inducibly bind and phosphorylate K protein may
serve not only to alter the activity of K protein itself, but K protein
may also provide an avenue for PKC
to engage in a cross-talk with
other K protein molecular partners in response to specific changes in
the extracellular environment.
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ACKNOWLEDGEMENTS |
---|
We thank Melissa Berry for carrying out
initial pilot experiments. We thank Dr. Uwe Rosenberger for the
purified mixture of bovine brain PKC,
, and
, Drs. James Watts
and Rudy Aebersold for baculovirus Lck, and Drs. Weiqun Li and Jacalyn
H. Pierce for HA-PKC
in pCEV27 expression vector.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM45134 and DK45978 and funds from the American Diabetes Association and the Northwest Kidney Foundation.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.
¶ Supported by a Yamagiwa-Yoshida Memorial International Cancer Study Grant from the International Union Against Cancer.
** Supported by a fellowship from the American Heart Association, Washington Affiliate.
To whom correspondence should be addressed: Dept. of Medicine,
Box 356521, University of Washington, Seattle, WA 98195. Tel.: 206-543-3792; Fax: 206-685-8661; E-mail:
karolb{at}u.washington.edu.
2 J. Ostrowski, D. Schullery, M. Shnyreva, O. Denisenko, H. Suzuki, M. Gschwendt, and K. Bomsztyk, manuscript in preparation.
3 D. S. Schullery, J. Ostrowski, O. N. Denisenko, L. Stempka, M. Shnyreva, H. Suzuki, M. Gschwendt, and K. Bomsztyk, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: hnRNP, heterogeneous nuclear ribonucleoprotein; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; IP, immunoprecipitation; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; a.a., amino acids.
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REFERENCES |
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