From the Massachusetts General Hospital Cancer Center and
Harvard Medical School, Charlestown, Massachusetts 02129 and
Dana-Farber Cancer Institute, Boston, Massachusetts
02115
Received for publication, September 4, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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A novel murine membrane-associated protein
kinase, PKK (protein kinase C-associated
kinase), was cloned on the basis of its physical
association with protein kinase C The protein kinase C
(PKC)1 family is made up of
at least 11 phospholipid-dependent serine/threonine kinases
(1-4). PKC was originally described as a calcium-activated,
phospholipid-dependent cytosolic serine/threonine kinase.
Conventional PKCs include PKC PKC We describe here a novel protein kinase that can physically associate
with PKC Plasmids--
HA-tagged PKC A Yeast Two-hybrid Assay for Proteins That Interact with the
Catalytic Domain of PKC In Situ Hybridization--
Whole mount in situ
hybridization on 8.5-10.5-day post coitum (dpc) WB mouse
embryos was carried out as described (19). In situ
hybridization on frozen sections of paraformaldehyde-fixed mouse
embryonic tissues with digoxigenin-labeled antisense probes was carried
out essentially as described by Ma et al. (20). Photomicrographs of whole mount embryos or sections following in
situ hybridization or immunolabeling were collected on an Olympus SZH10 stereo dissecting microscope or Nikon E600 compound microscope fitted with a SPOT I digital camera (Diagnostic Imaging).
Digoxigenin-labeled PKK,
PKC Transfections, Immunoprecipitation, and Kinase Assays--
293T
cells were transiently transfected with different plasmid constructs
using the calcium phosphate precipitation method as described (21).
Harvested cells were washed once with cold PBS and lysed in lysis
buffer (1 ml of 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM
Expression and Phosphorylation of GST Fusion Proteins--
GST
fusion proteins were isolated from BL21 cells transformed with GST
fusion expression plasmids according to the manufacturer's instructions (Amersham Pharmacia Biotech). Briefly, bacteria were initially grown at 37 °C for about 2-3 h
(A600 = 0.5-0.8) and subsequently
induced with 0.5 mM
isopropyl-1-thio-
For the phosphorylation assay, 1 µg of each purified fusion protein
was used as substrate for purified PKC Immunofluorescence and Fractionation of Membrane and
Cytosol--
293T or COS cells grown on coverslips and transfected
with Flag-tagged PKK constructs were fixed with 4% paraformaldehyde, permeabilized with 0.1% v/v Triton X-100 in PBS, and stained with anti-Flag antibody followed by fluorescein isothiocyanate-conjugated anti-mouse IgG.
In separate experiments, transfected cells were washed in PBS and were
allowed to swell on ice for 15 min in 20 mM Hepes, pH 7.4, containing 10 mM EDTA, 2 mM dithiothreitol, 10 µg/ml aprotinin, and 100 µM phenylmethylsulfonyl
fluoride. Cells were ruptured by repeated passage through a 27-gauge
needle. Nuclei were removed by centrifugation at 700 × g for 10 min. Membranes were isolated from the post-nuclear
supernatant by centrifugation at 75,000 rpm for 15 min on a Beckman
TL-100 Ultracentrifuge. The pellet (membranes) was taken up in 1%
(v/v) Triton X-100 in PBS, and Triton X-100 was also added to the
supernatant (cytosol) to bring it to 1% (v/v).
Metabolic Labeling and Pulse-Chase Studies--
Transfected 293T
cells were starved for 1 h in methionine-free medium and labeled
with 0.1-0.5 mCi of [35S]-methionine for 10 min. Cells
were either lysed immediately or chased for varying time periods with
an excess of complete medium. Lysates were immunoprecipitated as
described above.
Protein Phosphatase Treatment--
Phosphatase treatment of
immunoprecipitates and membrane pellets was carried out using
lambda protein phosphatase (New England Biolabs). Typically
400-1000 units of enzyme were used in a 100-µl suspension in 50 mM Tris-HCl, pH 7.5, containing 0.1 mM
Na2EDTA, 5 mM dithiothreitol, 2 mM
MnCl2, and 0.01% (v/v) Brij 35. Untreated and treated
samples were incubated at 30 °C for 30 min.
Identification of a Protein That Interacts with PKC PKK Is a Novel Ankyrin Repeat-containing Protein Kinase--
We
obtained a full-length clone from a murine embryonic cDNA library;
the sequence of this cDNA is depicted in Fig.
1a. The PKK
cDNA contains a 2358-base pair open reading frame with a
presumptive initiator methionine at nucleotide 50, a TAG stop codon at
nucleotide 2408, and an AAUAAA polyadenylation signal at nucleotide
3527, 11 base pairs upstream of the cleavage site. An in-frame stop codon was identified upstream of the first ATG codon. Preliminary analysis of the predicted protein sequence revealed a novel putative serine/threonine kinase with the kinase domain encompassing
approximately the first 300 amino acids. A stretch of 11 ankyrin
repeats was noted in the C-terminal half of the open reading frame
starting at residue 442 (schematically represented in Fig.
1c). In vitro transcription and translation in a
rabbit reticulocyte lysate yielded a protein with an apparent molecular
mass of 100 kDa (data not shown). Although no close homologs of
this cDNA were noted when this manuscript was originally prepared
for submission (see "Discussion"), an analysis of the 12 conserved
kinase subdomains suggested that it encoded a novel protein kinase. The
predicted protein encoded by this cDNA was called PKK (for
PKC-associated kinase). An examination of subdomains VI and VII
suggested that PKK represents a serine/threonine kinase with
conservation of a DLKPAN motif and a DFG triplet (23), but the
possibility that it represents a dual specificity serine/threonine and
tyrosine kinase was not excluded. The activation loop of PKK contains a Ser-X-X-X-Ser motif
characteristically found in MAP kinase kinases (24), which are
typically dual specificity kinases, and in related kinases such as IKK1
and IKK2 (25). Ten potential PKC phosphorylation sites, were noted and
these are underlined in Fig. 1a. A human PKK cDNA was
obtained using the mouse insert as a probe. Polymerase chain reaction
analysis of human/rodent radiation hybrids localized PKK to human
chromosome 21. The sequence of human chromosome 21 was published while
this manuscript was in preparation (26), and we confirmed the
localization of human PKK to chromosome 21q22.3. The complete sequence
of human PKK was confirmed from the human chromosome 21 sequence data
base (26).The mouse and human cDNAs encode almost
identical proteins, but the mouse sequence includes two residues
(Glu316 and Ser317) in the linker region
between the kinase domain and the ankyrin repeats that were not
predicted by the human sequence (Fig. 1b).
Northern blots (Fig. 2a and
data not shown) revealed that PKK is widely expressed in most tissues
but is expressed at very low levels in the spleen. PKK is, however,
relatively abundant in the thymus and is expressed in bone marrow in
pro-B, pre-B, and immature B cells (data not shown).
During embryogenesis, PKK mRNA transcripts were detected
at 10.5 dpc at diverse locations including the embryonic forebrain, otic vesicle, branchial arches, primitive gut, and genitourinary system
(Fig. 2b). Expression of
PKC
In Fig. 3, we provide formal evidence
that PKK is a protein kinase. In an immunoprecipitation kinase assay,
autophosphorylation of PKK was observed, and this kinase also
phosphorylates exogenous substrates such as myelin basic protein (data
not shown) and histone H1 (Fig. 3). These findings do not rule out the
possibility that an associated kinase (such as PKC PKK and PKC PKC
Since PKK apparently exists in multiple forms, we decided to examine
the subcellular localization of these distinct forms and to determine
whether their generation depended on post-translational modification events.
PKK Exists in Multiple Cytosolic and Membrane-associated
Forms--
When COS and 293T cells transfected with Flag-tagged PKK
were permeabilized and stained using indirect immunofluorescence, a
fine reticular staining pattern was observed, suggesting that PKK
associates with intracellular membranes (Fig.
6a). It was clear, however,
that PKK was not present in the nucleus. Examination of PKK by
subcellular fractionation (Fig. 6b) revealed that a major
portion of cellular PKK was cytosolic, the majority of this form being
seen as a 110-kDa protein. It is possible that a proportion of this
cytosolic protein is actually loosely associated with membranes in the
cell, in keeping with our immunofluorescence results. Extraction of
membranes with 1% Triton X-100 revealed no PKK, but two forms of PKK
were noted in the Triton-insoluble membrane fraction, one of 100 kDa
and another of 112 kDa (Fig. 6b). Because we were concerned
that a considerable amount of PKK may have been discarded in the
nuclear fraction, we examined the Triton X-100-extracted supernatants
and pellets of transfected 293T cells (Fig. 6c, left panel).
We also performed subcellular fractionation studies in which we
separated the cytosol from all pooled membrane fractions in the cell
including the membranes of the nuclear envelope. These separations were
performed both with 293T cells transfected with wild type PKK as well
as with cells transfected with K51R PKK (Fig. 6c, right
panel). As depicted in Fig. 6c, the major Triton X-100-soluble
form of PKK migrates at 110 kDa, and a minor amount of this
underphopshorylated 100-kDa form was also seen in the Triton X-100
supernatant. The soluble 110-kDa form corresponds to the 110-kDa form
seen in the cytosol (Fig. 6, b and c, right
panel). When detergent-resistant cell pellets were solubilized in
SDS loading buffer, a 112-kDa form and a 100-kDa form of PKK were
observed. Both the Triton X-100-resistant 112-kDa form and the 110-kDa
Triton X-100-extractable form of PKK yield a 100-kDa species when
subjected to lambda phosphatase treatment (Fig. 6d). There
are therefore three major forms of PKK that can be identified.
Most of the PKK in the cell is a soluble, primarily cytosolic or
loosely membrane-associated, phosphorylated 110-kDa form. A 100-kDa
under- or nonphosphorylated form of PKK represents a small proportion
of cytosolic PKK but makes up about one-half of the Triton
X-100-insoluble fraction of PKK. A 112-kDa hyperphosphorylated
detergent-insoluble form of PKK was also readily identified. It is
presumed that this protein is associated with cellular membranes
or with cytoskeletal elements. Although it is possible that a portion
of PKK may exist in detergent-insoluble glycolipid domains or lipid
rafts, immunofluorescence studies did not reveal discernible
accumulations of PKK on the inside of the plasma membrane in
transfected cells (Fig. 6a). In an attempt to obtain some
insights into the relationship between these different forms of PKK,
pulse-chase analyses of metabolically labeled PKK with and without
co-transfected PKC Conversion of PKK from an Underphosphorylated 100-kDa Species to a
110-kDa Phosphorylated Form Occurs Post-synthetically and Requires the
Catalytic Activity of PKK--
In transiently transfected cells
pulse-labeled with [35S]methionine for 10 min, wild-type
PKK is initially synthesized as a 100-kDa protein. In cells that have
been chased in complete medium for 20 min or longer, this 100-kDa
protein is quantitatively converted into a larger 110-kDa form.
Experiments in which PKK was examined after a 40-min or 1-h chase
period are depicted in Fig. 7a,
left panel, and b, respectively. A similar conversion
to the larger form has been noted after 20 min of chase, but only the
100-kDa form is seen after a 10-min chase period (data not shown). The post-synthetically derived larger form of PKK can be reduced to a
100-kDa form by treatment with lambda phosphatase, confirming that it
is derived by phosphorylation (Fig. 7a, right panel). A
kinase dead K51R mutant form of PKK is also initially synthesized as a
100-kDa form but is not converted to the 110-kDa form seen with wild
type PKK (Fig. 7b). Small amounts of slower migrating K51R
PKK proteins do appear with time; these might result from poorly
defined transphosphorylation events (Fig. 7b). PKK
phosphorylation and conversion to the 110-kDa form depends at least in
part on autophosphorylation. The delay in activating PKK suggests that the nascent enzyme might need to move to a specific location within the
cell or that it may in some temporally influenced manner interact with
an activating kinase or with some other regulator. We performed pulse-chase studies on PKK in cells co-transfected with PKC We have identified and molecularly cloned a novel serine/threonine
kinase, PKK, which physically associates with the catalytic domain of
PKC The cloning of a human ankyrin repeat-containing kinase based on its
association with PKC There is an apparent post-synthetic delay in the autophosphorylation of
PKK as suggested by pulse-chase studies. These results suggest that PKK
may need to interact with an activation loop kinase or some other
regulatory molecule in the cell, or to alter its subcellular
localization, in order to be activated. Because the N-terminal
catalytic domain of PKK is not phosphorylated by PKC The significance, if any, of the 112-kDa
hyperphosphorylated, Triton X-100-resistant form of PKK is unclear.
This fraction may potentially represent proteins that are in lipid
rafts or proteins that are tightly bound to cytoskeletal components.
The availability of antibodies that can be used to immunoprecipitate endogenous PKK would facilitate the examination of the biological significance of this form of PKK.
The expression of PKK is very tightly regulated during development.
This speaks to an important developmental role for this kinase. In
preliminary studies in which we targeted PKK K51R to developing
lymphocytes, early B and T cell development is significantly compromised.2 The defect in B
cell development observed in these preliminary studies is quite
distinct from the peripheral B cell defect seen in mice lacking PKC (PKC
). The regulated expression
of PKK in mouse embryos is consistent with a role for this kinase in
early embryogenesis. The human homolog of PKK has over 90% identity to
its murine counterpart, has been localized to chromosome 21q22.3,
and is identical to the
PKC
-interacting kinase, DIK
(Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and
Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a
C-terminal region containing 11 ankyrin repeats. PKK exhibits protein
kinase activity in vitro and associates with cellular
membranes. PKK exists in three discernible forms at steady state: an
underphosphorylated form of 100 kDa; a soluble, cytosolic,
phosphorylated form of 110 kDa; and a phosphorylated,
detergent-insoluble form of 112 kDa. PKK is initially synthesized as an
underphosphorylated soluble 100-kDa protein that is quantitatively
converted to a detergent-soluble 110-kDa form. This conversion requires
an active catalytic domain. Although PKK physically associates with
PKC
, it does not phosphorylate this PKC isoform. However, PKK itself
may be phosphorylated by PKC
. PKK represents a developmentally
regulated protein kinase that can associate with membranes. The
functional significance of its association with PKC
remains to be ascertained.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, PKC
I, PKC
II, and PKC
. They
require phosphatidylserine, diacylglycerol, and Ca2+
for maximal activation. PKC subfamilies exist which either do not
require calcium (novel PKCs) or which require neither calcium nor
diacylglycerol for optimal activation (atypical PKCs). Specific PKC
isozymes play important and distinct roles in the development and
activation of most vertebrate cell types including lymphocytes, but the
study of the function of individual isozymes is in its infancy.
I and PKC
II are generated by alternative splicing at the
PKC
locus and differ over a 50-52-amino acid stretch at their C
termini. This locus has been shown to be critical for the generation of
B-1 B lymphocytes and for the activation of peripheral B cells by
T-independent antigens (5). PKC
has also been shown to be required
in HL-60 cells for phorbol 12-myristate 13-acetate-induced differentiation into macrophages (6, 7). One of the best studied
substrates of PKCs in general, MARCKS (myristoylated alanine rich
protein kinase C substrate (8)) is also a substrate of PKC
(9). A
number of other proteins shown to be phosphorylated by PKC
I and/or
PKC
II include LIM domain proteins (10), Cut homeodomain
proteins (11), tyrosinase (12), nuclear lamins (13), PKC-interacting
protein-1 (14), and F-actin (15). Although a number of proteins that
are phosphorylated by PKC
have been described, the substrate(s) of
PKC
critical for B cell development remains to be identified.
. We show that this novel ankyrin repeat-containing kinase,
which we call PKK (for PKC-associated kinase), is associated with
membranes, is initially synthesized as a soluble 100-kDa protein, and
is subsequently converted to a larger phosphorylated form. This
conversion depends, at least in part, on the intrinsic catalytic
activity of this kinase. At steady state, a portion of this protein is
membrane-associated. The significance of the association of PKK with
PKC
remains to be determined.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
I, PKC
, and PKC
plasmids
were generous gifts from Dr. Shun'ichi Kuroda (10), Dr. Peter Parker
(16), and Dr. Weiqun Li (17), respectively. The kinase domains of
PKC
I (residues 291-673), PKC
II (residues 291-673), and PKC
(residues 343-674) were used as baits in a yeast two-hybrid approach
and were individually cloned into the SalI/SpeI
sites of pPC97AD (Life Technologies, Inc.; kindly provided by Dr. Marc
Vidal, Dana-Farber Cancer Institute). A kinase dead PKK K51R mutant,
was created using polymerase chain reaction-based mutagenesis and
confirmed by sequencing. In-frame glutathione S-transferase
(GST) fusion constructs involving distinct fragments of PKK were
generated using pGEX2T (Amersham Pharmacia Biotech).
1--
A yeast two-hybrid screen was
performed as described (18). A pPC86DB-cDNA library was made with
12-13-day whole murine fetuses (gift of Dr. Joshua LaBaer, Harvard
Medical School). The expression of bait plasmids was confirmed by
Western blotting using individual isotype-specific antibodies. For
library screening, the yeast MaV103 strain carrying the
pPC97AD-PKC
1-(291-673) plasmid was transformed with the pPC86DB
cDNA library. Transformants were replica-plated onto different
selective plates as described (18). The specificity of cDNAs
obtained from positive yeast colonies was analyzed by retransformation
with different controls. Both strands of a 3.4-kilobase pair
full-length cDNA clone obtained from the two-hybrid assay were
sequenced, and this insert was cloned into a pCMV-driven expression
vector with a Flag tag.
I, and PKC
II
probes were used in these studies.
-mercaptoethanol, 50 mM NaF, 1 mM
Na3VO4, 1 mM phenylmethysulfonyl
fluoride, 10 µM pepstatin, and 1% (v/v) Triton X-100).
Nuclei were removed by centrifugation, and the cell lysates were
incubated for 30 min on ice with either 3 µg of anti-Flag (M2,
Eastman Kodak Co.) or anti-HA (12CA5, Roche Molecular Biochemicals)
monoclonal antibody followed by incubation with 10 µl of protein
G-Sepharose 4 fast flow (50% slurry, Amersham Pharmacia Biotech) at
4 °C for 1 h. The beads were washed four times with lysis
buffer and twice with kinase buffer (see below). Half of the beads were
reserved for Western blotting. The other half of each sample was used
in an in vitro kinase assay. Beads were suspended in 50 µl
of kinase buffer containing 20 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM CaCl2, 8 µg/ml phosphatidylserine, and 0.8 µg/ml diolein (Sigma). After the
addition of 1.0 µl of [
-32P]ATP (10 mCi/ml), the
reaction mixture was incubated for 30 min at 30 °C. The reaction was
stopped by the addition of SDS loading buffer. Samples were analyzed on
an 8% polyacrylamide-SDS gel. The gel was dried, and proteins were
visualized by autoradiography.
-D-galactopyranoside for 3-4 h. Cells
were sonicated, and the fusion proteins were purified on
glutathione-agarose beads (Amersham Pharmacia Biotech). Purified GST
fusion proteins were dialyzed in kinase buffer or PBS (for the binding
assay), respectively, and protein concentrations were measured. GST
fusion proteins were analyzed by immunoblotting using anti-GST antibodies.
1(Calbiochem) in 40 µl of
the kinase buffer (described above). 1 µl of
[
-32P]ATP (10 mCi/ml) was added, and the mixture was
incubated at 30 °C for 30 min. The reaction was stopped by adding
SDS loading buffer; samples were run on SDS-polyacrylamide gels,
and proteins were visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
A
two-hybrid screen was initiated using the catalytic domain of PKC
I
as a bait. Our initial goal was either to identify targets of PKC
or
to identify proteins that might regulate the activity of PKC
. In
particular we were aware that we might identify protein kinases, other
than PDK1 (22), required for the activation of PKC
. The yeast strain
used in this two-hybrid assay contains three stably integrated reporter
constructs expressing HIS3, LacZ, and URA3. Clones were
considered to have scored positively if they grew on plates lacking
histidine and containing 3-AT, if they turned blue on
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal)
plates, and if they grew on plates lacking uracil but failed to grow in
5-fluoroorotic acid (5-FOA), which is toxic if URA3 is expressed.
4 × 106 independent transformants from a mouse embryo
library were screened using this stringent approach. Only one positive
clone was obtained. Rescreening with specific baits revealed that the
product of this clone interacted specifically with the catalytic domain
of PKC
I but not with a number of other baits including the catalytic
domain of PKC
II, the Rb-related p130 pocket protein, the cytoplasmic tail of CD44, or the DP1 subunit of E2F·DP complexes.
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Fig. 1.
PKK is a novel ankyrin repeat-containing
kinase. a, nucleotide and deduced amino acid
sequences of mouse PKK cDNA. Nucleotide and amino acid
residues are numbered on both sides. The 10 potential PKC phosphorylation sites are underlined. The
GenBank accession number is AF302127. b, alignment of
peptide sequences of mouse and human PKK. A sequence comparison was
carried out using the GeneWorks program. Amino acid residues are
numbered on the right side. Identical residues
are boxed. Residues Glu316 and
Ser317 were not noted to be encoded by the human sequence.
c, a schematic representation of PKK.
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Fig. 2.
The expression of PKK is
developmentally regulated. a, Northern blot analysis of
PKK mRNA. A blot of polyadenylated mRNA isolated from various
adult mouse tissues (CLONTECH) was used. Only a
single 4.4-kilobase transcript of PKK was detected (upper
panel). The blot was reprobed with mouse -actin (lower
panel). b
g, analysis of PKK,
PKC
I, PKC
II expression by in
situ hybridization in developing mouse embryos. b
d,
mouse embryos at 10.5 dpc were subjected to whole mount in
situ hybridization. b, early expression of
PKK in the developing telencephalon (t),
diencephalon (di), otic vesicle (ot), branchial
arches, gut (gt), and genitourinary systems. The
closed arrow indicates expression in the primitive vessels,
and the open arrow shows expression in the branchial arches.
c and d, expression of
PKC
I (c) and
PKC
II (d) overlapped that of
PKK in the primitive vessels (closed arrow) and
the branchial arch primordium (open arrow). Note that a
scattered population of PKC-expressing cells was present in
the spinal cord. e, at 12.5 dpc, ongoing expression in the
gut (gt) and genitourinary system (e.g. genital
tubercle (ge)) is detected, as well as transient expression
in the ventral neural tube (arrow). f and
g, at 14.5 dpc, strong expression throughout the
gastrointestinal tract is detected (es, esophagus;
du, duodenum; mg, midgut). The arrow
in panel f indicates expression in the skin.
I and PKC
II
(Fig. 2, c and d) broadly mimicked that of
PKK in the primitive vessels and branchial arch primordium,
although PKK and PKC
expression only partially overlap during development. At 12.5 dpc, ongoing expression was detected in the gut, the mesonephric duct of the genitourinary tract, and the urogenital sinus. Interestingly, we noted transient expression of PKK in the ventral neural tube at 12.5 dpc
(arrow, Fig. 2e) but not at the earlier or later
stages tested. By 14.5 dpc, strong expression throughout the
gastrointestinal tract was observed in the luminal tissues of the
esophagus, stomach, duodenum, and intestines (Fig. 2, f and
g), as well as transient expression in the skin. In
addition, PKK was expressed in the collecting system of the
genitourinary tract but not in kidney. These results are consistent
with the possibility that PKK has roles during embryonic development.
) may mediate some
of these activities. However, the PKK K51R mutant (kinase dead PKK) has limited immunoprecipitation kinase activity, indicating that PKK is
indeed a kinase (Fig. 3). PKK is frequently visualized in transfected cells as a doublet in the 100-110 kDa range (see Figs.
4-7). The relationship between some of
the different forms of this protein has been established and is
described below (see Figs. 6 and 7).
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Fig. 3.
PKK is a protein kinase. An in
vitro immunoprecipitation kinase assay was carried out as
described under "Experimental Procedures." 293T cells were
transfected with vector alone or Flag-tagged wild type or kinase dead
PKK K51R as indicated. Cleared lysates were immunoprecipitated using an
anti-Flag antibody. Histone H1 was used as substrate. A portion of each
lysate was subjected to an anti-Flag Western blot analysis (lower
panel).
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Fig. 4.
PKK interacts with
PKC I. HA-tagged PKC
I was
co-transfected with vector alone or with Flag-tagged wild type PKK or
PKK K51R into 293T cells. Cell lysates were immunoprecipitated either
with an anti-Flag antibody or with anti-HA antibody. The
immunoprecipitates (IP) were analyzed by
immunoblotting (WB, Western blot) with anti-HA or anti-Flag
antibodies. An aliquot (one-tenth of the total cell lysate) was
used for immunoblotting with the corresponding anti-tag antibodies as
indicated.
Can Physically Associate with One
Another--
We have confirmed that PKK and PKC
can
associate with each other in vivo. Flag-tagged PKK and
HA-tagged PKC
1 were co-transfected into 293T cells. As seen in Fig.
4, reciprocal immunoprecipitations and Western blot analyses reveal
that PKK and PKC
associate with one another.
I Can Phosphorylate PKK in Vitro--
We have demonstrated,
using immunoprecipitated PKK and a fusion protein in which a portion of
maltose-binding protein was fused to kinase dead PKC
1, that PKK
cannot phosphorylate PKC
I in vitro (data not shown). We
therefore consider it extremely unlikely that PKK is required for
PKC
activation. We wished to establish whether PKC
1
phosphorylates PKK in vitro. We transfected 293T cells with
Flag-tagged PKK K51R and used an anti-Flag immunoprecipitate as the
substrate in an in vitro kinase assay with purified PKC
I (Calbiochem). As seen in Fig.
5a, PKK K51R is readily
phosphorylated by PKC
I. As depicted in Fig. 5b, purified
GST fusion proteins including C-terminal portions of PKK
(residues 402-786 and 461-786) were phosphorylated in
vitro, whereas a fusion protein including the N-terminal portion
(residues 1-320) was not. However, in numerous experiments in which
PKK was coexpressed with PKC
I or with PKC
, we were unable to
demonstrate a reproducible and significant enhancement of the catalytic
activity of PKK by either of these PKCs (data not shown).
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Fig. 5.
Phosphorylation of PKK by PKC I.
a, PKC
I can phosphorylate PKK in vitro.
Lysates from 293T cells transfected with PKK K51R were precipitated
with anti-Flag antibody. Half of the immunoprecipitate was incubated
with [
-32P]ATP and was used as the substrate in an
in vitro kinase assay using purified PKC
1. The other half
was also incubated with [
-32P]ATP in the absence of
added PKC
1. One-tenth of each cell lysate was set aside for a
Western blot with anti-Flag antibody. b, the C-terminal
portion of PKK can be phosphorylated by PKC
I in vitro.
Purified GST-PKK fusion proteins as indicated in the figure were used
as substrates for purified PKC
I. An in vitro kinase assay
was performed as described under "Experimental Procedures." Samples
were separated on an 8% polyacrylamide-SDS gel. In the lower
panel, individual fusion proteins were separated on a 12%
polyacrylamide-SDS gel and stained with Coomassie Blue.
were undertaken.
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Fig. 6.
PKK exists in multiple underphosphorylated
and phosphorylated forms. a, COS cells were transfected with
Flag-tagged wild type PKK, and permeabilized cells were stained with
anti-Flag monoclonal antibodies and fluorescein isothiocyanate-anti
mouse IgG. b, subcellular fractionation of 293T cells
expressing wild type PKK. Cytosol was obtained from the post-nuclear
supernatant of disrupted cells by ultracentrifugation. Membranes were
extracted (TSM, Triton-soluble membranes) with 1% Triton
X-100 in PBS. The remaining pellet was solubilized using SDS sample
buffer (TIM, Triton-insoluble membranes). 4% of the total
cytosol was loaded in the first lane, whereas 25% of the TSM and TIM
were loaded in the adjacent lanes. An anti-Flag Western blot assay was
performed. c, left panel, 293T cells transfected
with wild type (WT) PKK were lysed with 1% Triton X-100;
25% of the pellet and 2.5% of the supernatant (Lysate)
were separated by SDS-polyacrylamide gel electrophoresis, and
Flag-tagged PKK was visualized on a Western blot. Right
panel, subcellular fractionation of 293T cells transfected with
wild type or K51R PKK. Cells were separated into a cytosolic fraction
and a pellet, which included all membranes as well as the nuclear
pellet. 2% of the cytosol and 50% of the pellet were separated by
SDS-polyacrylamide gel electrophoresis, and Flag-tagged PKK was
visualized on a Western blot. d, a 1% Triton X-100 lysate
of 293T cells transiently transfected with wild type PKK was
immunoprecipitated, and half of the immunoprecipitate (IP)
was subjected to lambda phosphatase treatment. A portion of the pellet
was also treated with lambda phosphatase (left panel).
Another experiment in which equivalent amounts of phosphatase-treated and untreated
pellets were separated is depicted in the right panel. PKK
was revealed by an anti-Flag Western blot.
I, and
found that even with the co-expression of PKC
I, a similar delay was
observed in the post-synthetic generation of the 110-kDa form of PKK
(data not shown). Although PKK probably requires activation by a
distinct regulator, which is most likely an activation loop kinase,
overexpression of PKC
I does not influence the in vivo generation of the 110-kDa form of PKK.
View larger version (31K):
[in a new window]
Fig. 7.
Post-synthetic generation of a phosphorylated
form of PKK. a, pulse-chase analysis of transiently
transfected 293T cells reveals that PKK is synthesized initially as a
100-kDa protein, which is subsequently converted into a slower
migrating 110-kDa form (left panel). In a separate
experiment, cells were labeled metabolically with
[35S]methionine for 10 min. After a 1-h chase, PKK was
immunoprecipitated, and one-half of the precipitate was treated with
lambda phosphatase (right panel). b, pulse-chase
analysis of cells transiently transfected with wild type PKK or with
the K51R mutant. See the legend to panel a and the
text for details. A prominent background band that migrates
faster than PKK is seen in panel a and
also in panel b. It is seen in anti-Flag immunoprecipitates
of metabolically labeled mock-transfected 293T cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. This interaction was observed in the context of a two-hybrid
assay using recombinant fusion proteins in vitro and in
transfected cells in vivo. The interaction in the two-hybrid system suggests that PKC
I and PKK interact directly, and it is possible that PKK may represent a substrate of PKC
and also of other
members of the PKC family. The C-terminal portion of PKK is
phosphorylated by PKC
I in vitro. We have no evidence to
suggest that PKK is actually phosphorylated by PKC
I in
vivo. We have also been unable to demonstrate that PKC
I
influences the catalytic activity of PKK.
has been also recently described (27). This
kinase, DIK (for PKC
-interacting kinase), is identical to human
PKK. DIK was shown to interact with the catalytic domain of PKC
, but
PKC
does not phosphorylate DIK. It is unclear whether there is any
physiological relevance to the interaction of PKK with PKC
or
PKC
. Although there is some overlap in the expression patterns of
PKC
and PKK during embryogenesis, there are clearly developing
tissues in which PKK is expressed in the absence of PKC
. It is
formally possible that different members of the PKC family might
regulate PKK activity in different cell types. At this time it is not
possible to functionally link PKK to PKC
or to any other member of
the PKC family.
I, it is likely
that PKC
I does not directly influence the phosphorylation of the
activation loop of PKK. The activation loop of PKK contains a
Ser-X-X-X-Ser motif, which suggests
that PKK is related to MAP kinase kinases and that it may be also
activated in vivo by a MAP kinase kinase kinase. Studies are
currently in progress to identify the MAP kinase kinase kinase that may
be required for the activation of PKK.
(5). Studies are in progress to determine whether this kinase plays a
role in early embryonic development.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. L. Aravind for pointing out the
11th ankyrin repeat and Dr. C. Ashendel for the
PKCII probe.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants AI 33507 and CA 69618 from the National Institutes of Health. This work was presented in preliminary form at the 10th International Congress of Immunology, November 1998, New Delhi, India, and in abstract form (Parng, C., Chen, L., Cariappa, A., Sabatos, C., Ponda, M., Kim, T. J., and Pillai, S. (1998) Immunologist Suppl. 1, p. 383).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF302127.
§ To whom correspondence should be addressed: MGH Cancer Center, Bldg. 149, 13th St., Charlestown, MA 02129. Tel.: 617-726-5619; Fax: 617-724-9648; E-mail: pillai@helix.mgh.harvard.edu.
Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M008069200
2 L. Chen, A. Cariappa, K. Haider, M. Tang, and S. Pillai, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; PKK, PKC-associated kinase; GST, glutathione S-transferase; dpc, days post coitum; PBS, phosphate-buffered saline; MAP, mitogen-activated protein; Lin-11, Isl-1, and Mec-3.
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REFERENCES |
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