(Received for publication, June 19, 1995; and in revised form, September 4, 1995)
From the
The heterogeneous ribonucleoprotein particle (hnRNP) K protein interacts with multiple molecular partners including DNA, RNA, serine/threonine, and tyrosine kinases and the product of the proto-oncogene, Vav. The K protein is phosphorylated in vivo and in vitro on serine/threonine residues by an interleukin 1 (IL-1)-responsive kinase with which it forms a complex. In this study we set out to map the K protein domains that bind kinases. We demonstrate that the K protein contains a cluster of at least three SH3-binding sites (P1, PPGRGGRPMPPSRR, amino acids 265-278; P2, PRRGPPPPPPGRG, 285-297; and P3, RARNLPLPPPPPPRGG, 303-318) and that each one of these sites is capable of selectively engaging c-Src and Vav SH3 domains but not SH3 domains of Abl, p85 phosphatidylinositol 3-kinase, Grb-2, and Csk. We demonstrate that the K protein domain that recruits and is phosphorylated in an RNA-dependent manner by the IL-1-responsive kinase, designated KPK for K protein kinase, is contained within the 338-425-amino acid stretch and thus is contiguous but does not include the cluster of the SH3-binding sites. K protein and KPK co-immunoprecipitate from cell extracts with either c-Src or Vav, suggesting that K protein-KPK-c-Src and K protein-KPK-Vav complexes exist in vivo. Furthermore, in the context of K protein, c-Src can reactivate KPK in vitro. The succession of kinase-binding sites contained within the K protein that allow it to form multienzyme complexes and facilitate kinase cross-talk suggest that K protein may serve as a docking platform that promotes molecular interactions occurring during signal transduction.
The heterogeneous ribonucleoprotein particle (hnRNP) ()K protein is endowed with diverse biochemical properties,
and, as a result, the K protein is a highly interactive molecule. K
protein was first discovered as a component of hnRNP, where it exists
bound to RNA(1, 2) . There are several isoforms of K
protein that represent alternatively spliced products of a gene that in
humans has been mapped to chromosome 9(3) . K protein binds
preferentially to poly(C) compared to other ribonucleic acid
homopolymers(1, 2) . As a component of the hnRNP, K
protein may participate in processing and transport of
pre-mRNA(4) . The hnRNP contains at least 20 major proteins
that have different RNA binding specificities. It is already clear that
the differences between K protein and the other hnRNP proteins go far
beyond differences in RNA binding properties. K protein also binds
single- and double-stranded DNA in a sequence-selective manner and can
activate gene promoters that contain cognate DNA
motifs(5, 6, 7) . In these two respects, the
K protein resembles the Xenopus transcription factor TFIIIA;
TFIIIA is both a positive transcription factor and serves to store and
transport 5 S RNA to the cytoplasm(8, 9) .
The functional diversity of the K protein appears to be even greater than that described for TFIIIA. The K protein was recently discovered to bind the SH3 domains of the protein-tyrosine kinases, Src, Fyn, and Lyn(10, 11) . Although these observations suggested that K protein can potentially engage these protein-tyrosine kinases, it is not itself tyrosine-phosphorylated(10, 12) . Nonetheless, the potential interaction with these tyrosine kinases suggests that the K protein is involved not only in the regulation of gene expression but also in signal transduction. A link to the signal transduction cascade is further illustrated by recent reports that K protein interacts in vivo and in vitro with the proto-oncogene product, Vav (13, 14) . The interaction of K protein with Vav is mediated via SH3 domains, another typical feature of signal transduction processes (10, 11, 13, 14) . The K protein potential to interact with c-Src and its ability to bind RNA resembles the property of another protein, p68/p62 or Sam68, that is a target for c-Src and also binds RNA(11, 15, 16) .
K
protein is phosphorylated in vivo and in vitro on
serine and threonine residues(12) . At least, in part, this
phosphorylation is mediated by an associated kinase which can respond
to treatment of cells with IL-1 and other
agents(12, 17) . This IL-1-responsive kinase adds yet
another factor to the list of K protein molecular partners. The
identity of this kinase is not known(12, 17) . This
enzyme is activated by phosphorylation (17) and might,
therefore, be linked to one of the kinase cascades(18) .
Phosphorylation of K protein by K protein kinase (KPK) is facilitated
by either the B enhancer or poly(C) RNA motifs, both of which can
bind the K protein (6, 12, 17) . Whether
other DNA enhancer motifs that recognize K protein (5, 7) influence its phosphorylation is not known.
Because K protein interacts with some of its molecular partners via SH3
interactions(10, 11, 13, 14) , and
the fact that serine/threonine kinases that contain SH3 domains have
been identified(19) , we set out to map the K protein
SH3-binding domains and determine whether or not these sites
participate in the recruitment of KPK to K protein.
To obtain GST-Src SH3 protein labeled with P,
Src SH3 fragment was cloned into pGEX-2TK vector (Pharmacia, Uppsala,
Sweden) (23) using BamHI and PstI sites.
After transformation of BL21(DE3)pLysS cells with this plasmid, GST-Src
SH3 protein was expressed as described above. After binding to
glutathione beads, labeling was done directly on the beads using
[
-
P]ATP and protein kinase A as per
manufacturer's protocol (Pharmacia).
P-Labeled
GST-Src SH3 protein was eluted from the beads with 5 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0.
Figure 1:
Far
Western mapping of the Src SH3-binding domain in K protein. 5 µg of
purified GST-fusion proteins containing deletion fragments of K protein
K13, aa 1-337 (lane 1); K10, aa 318-382 (lane
2); K7, aa 318-464 (lane 3); K6, aa 84-209 (lane 4); K5, aa 171-464 (lane 5); K3, aa
171-337 (lane 6); K2, aa 84-337 (lane 7);
and K1, aa 425-464 (lane 8), or the full-length K
protein, aa 1-464, (lane 9) were separated by SDS-PAGE
and were electrotransferred to Immobilon-P membrane. After
electrotransfer, blots were either immunostained with anti-GST antibody
(immunoblot, A) or probed with P-labeled
GST-SrcSH3 fusion protein (autoradiograph, B). The GST-fusion
protein constructs and the localization of the K protein stretch that
contains the Src SH3-binding domain (Src SH3-BD) is illustrated in the bottom panel. Molecular mass markers are shown in kDa.
Predicted sizes (in kDa) are: GST-K, 91.0; GST-K1, 30.4; GST-K2, 53.9;
GST-K3, 42.2; GST-K5, 56.2; GST-K6, 39.8; GST-K7, 42.3; GST-K10, 33.1;
GST-K13, 60.1.
Although the
intensities of the major immunostained bands for each of the GST-K
deletion mutants were approximately the same (Fig. 1A),
the levels of the P signals were quite different (Fig. 1B). For example, the
P signal of
the bands that correspond to the GST-K3 (aa 171-337, lane
6) and GST-K2 (aa 82-337, lane 7) deletion mutants
is stronger than the
P intensity seen with the longer
GST-K13 (aa 1-337, lane 1) mutant. This observation
suggests that the affinity of the shorter mutants which recognize the
c-Src SH3 domain is higher. This might explain the equal signal seen
with the full-length GST-K protein (90 kDa) and the less abundant
fragment of 60 kDa co-expressed in that preparation (Fig. 1,
compare lanes 9 in A and B). The reason for
the apparently lower SH3 binding affinities of the longer GST-K protein
fragments may reflect a higher degree of masking of the SH3-BD when
this domain is present in the context of a larger protein.
Next we
tested K protein specificity as a ligand for SH3 domains from different
proteins involved in signal transduction. Far Western blot analysis
from this experiment is illustrated in Fig. 2. Immunoblotting
with the anti-K antibody No. 54 shows the position of the K protein (lane 1). As before (Fig. 1), the GST-Src SH3 domain
bound to K protein when directly used as a probe (lanes 8). In
contrast, SH3 domains from the phosphatidylinositol 3-kinase-associated
p85 subunit (26) (lane 2), Ras-GAP (27) (lane 3), Grb2 (28) (lane 4,
NH terminus SH3, and lane 5, COOH terminus SH3),
Abl (29) (lane 6), and c-Src kinase (Csk) (lane
7) (11) did not bind to the K protein. These results show
that the ability of K protein to bind SH3 domains is selective for the
c-Src SH3 domain.
Figure 2:
Far Western blot analysis of K protein
probed with different SH3 domains. Purified nuclear K protein was
electrophoresed on SDS-PAGE and transferred to Immobilon-P membrane.
Each lane contained the same amount of K protein. K protein in lane
1 was identified with anti-K antibody (antibody No. 54 (17) at 1:10,000 dilution). Proteins in lanes 2-8 were probed with a panel of GST SH3 domains (2 mg/ml) from
phosphatidylinositol 3-kinase-associated p85 subunit (26) (lane 2), Ras-GAP (27) (lane 3),
Grb-2 (28) (lane 4, NH terminus SH3, and lane 5, COOH terminus SH3), Abl (29) (lane
6), c-Src kinase (Csk) (lane 7)(11) , and c-Src
(Src) (lane 8). The GST fusion proteins were detected with
anti-GST monoclonal antibody (0.1 mg/ml). Molecular mass markers are
shown in kDa.
Figure 3: Binding of the GST-Src SH3 and GST-Csk SH3 fusion proteins to synthetic peptides representing different K protein domains. Peptides, P1-P5, were synthesized based on the K protein amino acid sequence and covalently conjugated to SulfoLink Gel Beads (see ``Materials and Methods''). Beads bearing the specific synthetic peptides were saturated with BSA (25 mg/ml) by overnight incubation (4 °C). After a four-time wash with 1 ml of binding buffer, beads were incubated for 30 min at 4 °C in 1 ml of binding buffer containing either Src- or Csk-GST SH3 fusion protein (2 mg/ml). After binding, beads were spun down, supernatants were saved, and beads were washed four times with 1 ml of binding buffer containing 0.1% Triton X-100 and 0.2% deoxycholate. Proteins bound to the beads were eluted by boiling in loading buffer, while proteins in the supernatants were first precipitated with acetone (45) and then boiled in loading buffer. Proteins precipitated by the beads (A, lanes 7-12, and B, lanes 1-6), and those remaining in the supernatants (A, lanes 1-6) were analyzed by SDS-PAGE and immunoblotting with an anti-GST monoclonal antibody (0.1 mg/ml). The amino acid sequences used to design the synthetic peptides and their location in the K protein are shown below the blots.
As with
c-Src, K protein has also recently been shown to engage Vav through SH3
interactions(13, 14) . Vav contains two SH3 domains,
but only the one present in the COOH terminus (Vav SH3C) binds K
protein (13) . We used the same approach with the
peptide-bearing beads to determine which of the K protein proline-rich
domains can engage Vav SH3 domain. Results of this experiment are shown
in Fig. 4. As before (Fig. 3), proteins in the
supernatant and proteins bound to the peptide beads were analyzed by
SDS-PAGE and immunoblotting using anti-GST antibody. The results showed
that beads bearing each one of the proline-rich peptides (P1, P2, or
P3) precipitated most of the GST-Vav SH3C fusion protein (compare beads (lanes 8-10) to supernatant (lanes 3-5)).
The P3 peptide appeared most efficient since, after mixing with these
beads, there was no GST-Vav SH3C protein remaining in the supernatant
(compare lane 3 to lanes 4 and 5). In
contrast to the beads bearing the proline-rich peptides, with beads
bearing P4 (aa 1-12) and P5 (aa 50-60) peptides, most of
GST-Vav SH3C remained in solution (lanes 1 and 2),
with only a trace amount of the protein bound to the beads (lanes 6 and 7). No binding was observed with GST-Vav SH2 and the
NH terminus GST-Vav SH3 domains to any of the peptide beads
(data not shown). Therefore, as with c-Src SH3 domain (Fig. 3),
each one of the K protein proline-rich sites can independently and
specifically bind the Vav COOH terminus SH3 domain. Because there was
significantly less GST-Vav SH3C protein remaining in the supernatant
from beads bearing P3 peptide (compare lane 3 to lanes 4 and 5), the Vav SH3C domain exhibits preference for the
third SH3-binding site, P3.
Figure 4: Binding of GST-Vav SH3 COOH terminus fusion protein to synthetic K protein peptides. The same protocol described in Fig. 3was used here. The GST-Vav COOH terminus SH3 (Vav SH3C) fusion protein that bound to the beads was run in lanes 6-10, supernatants from the same beads were run in parallel in lanes 1-5. Molecular markers are shown in kDa (lane 11).
Figure 5:
Comparison of affinities of the K protein
proline-rich sites for the c-Src SH3 domain. A, equal amounts
of purified GST-K protein (1 µg/lane) was electrophoresed on
SDS-PAGE and transferred to Immobilon-P membrane. After renaturation
and blocking, the membranes were mounted in a manifold device (Hoefer,
San Franscisco, CA), and each lane was incubated for 2 h (25 °C)
with 2 mg/ml P-labeled GST-Src SH3 probe alone (lane
2) or in the presence (lanes 3-9) of increasing
concentrations of either P1, P2, or P3 synthetic peptides. 750
µM P5 synthetic peptide was used as a negative control (lane 1). After binding, membranes were washed and
autoradiographed. B,
P-labeled bands were excised
from the membranes and counted in a scintillation counter. Binding of
the
P-labeled GST-Src SH3 probe to GST-K is expressed as
percent of maximal counts (100% = no peptide added, lane
2). C, amino acid sequences of the synthetic peptides
(P1, P2, P3, and P5) and their location in the K
protein.
For two
competitive inhibitors, the following relationship holds true,
`IC/"IC
=
`K
/"K
, where K
is an inhibitor dissociation constant for an
inhibitor(31) . Also, the c-Src SH3 domain interactions with
any of the two K protein proline-rich sites can be depicted by the
following, `K
/"K
`K
/"K
, where K
is the dissociation constant for a given site. Thus, using the
IC
ratios, we estimate that the relative affinity of the K
protein P3 site for the Src SH3 domain is about 5-fold higher than the
affinity of the P2 site and approximately 25-fold higher than the
affinity of the P1 proline-rich site. Collectively, the above studies (Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5)
suggest that (i) the K protein contains at least three regions that
bind the Src SH3 domain, (ii) each one of the regions binds c-Src SH3
domain with significantly different affinities, and (iii) the K
values of these interactions are in the
micromolar range.
Figure 6:
Mapping of the K protein domains that bind
and are phosphorylated by the K protein-associated kinase. 50 µl of
purified nuclear K protein/K protein-associated kinase preparation was
mixed overnight (4 °C) with glutathione beads (30-µl beads in 1
ml of binding buffer) bearing either the full-length or one of the
deletion fragments of K protein fused to GST. After binding, beads were
spun down and washed, and phosphorylation of the beads was done in 270
µl of 1 binding buffer/30 µl of 10
kinase
buffer (200 mM HEPES, pH 7.5, 100 mM
MgCl
, 50 µM dithiothreitol) containing 5
µCi of [
-
P]ATP with (+) or
without(-) poly(C) RNA. Phosphorylation was terminated by washing
the beads twice with 800 µl of binding buffer.
P-Labeled proteins were eluted from the beads and were
analyzed by SDS-PAGE and autoradiography. The lower panel illustrates the GST-K protein constructs used in the assay. Src SH3 BD, Src SH3-binding domain, KPK-BD, K protein
kinase-binding domain, and
P-ATP/binding designates the GST-K constructs that were phosphorylated by the
bound kinase in an RNA-dependent manner. Molecular mass standards are
shown in kDa. Predicted size (in kDa) for GST-K4 was 70 and for GST-K8,
37.5. (See Fig. 1legend for the size of the other
mutants.)
The fact that the GST-K7 deletion mutant (aa 318-464) was phosphorylated, while the GST-K1 deletion mutant (aa 425-464) was not, may indicate that either the K1 fragment does not engage KPK, that it does not contain suitable phosphorylation sites, or both. The next series of experiments was done to determine whether or not the K1 fragment can recruit KPK. Glutathione beads bearing plain GST, GST-K1, or GST-K7 fusion proteins were mixed with IgG-affinity-purified K protein/KPK preparation. Following binding, beads were spun down, and supernatants were then mixed with beads containing the full-length GST-K protein. Washed glutathione beads from the first and the second round of binding were phosphorylated as before with or without poly(C) RNA. Fig. 7illustrates Coomassie Blue-stained (upper part) and autoradiographed SDS-PAGE (lower part) of proteins eluted from these beads (Beads, left panel). As in Fig. 6, GST-K7 (Beads, lanes 5 and 6) was phosphorylated in an RNA-dependent fashion, while plain GST (lanes 1 and 2) and GST-K1 (lanes 3 and 4) were not phosphorylated at all. The supernatants (right panel) from plain GST (lanes 1 and 2) and GST-K1 (lanes 3 and 4) beads contained a kinase activity that bound and phosphorylated the full-length GST-K protein to a far greater extent and in an RNA-dependent manner compared to the supernatant from the GST-K7 beads (lanes 5 and 6). These results demonstrate that while glutathione beads bearing the GST-K7 deletion mutant removed KPK from the preparation, the GST-K1 fragment containing the last 40 COOH terminus K protein amino acid residues did not, indicating that the 425-464 region does not contain the kinase-binding domain. Thus, the KPK-binding domain is contained within the 318-425-amino acid segment of the K protein. This domain is adjacent to but does not include the cluster of the three SH3-binding sites (see the diagram at the bottom of Fig. 7), illustrating that KPK does not bind to K protein through SH3 interactions.
Figure 7:
Mapping of the K protein domain that binds
the K protein kinase, KPK. 30 µl of glutathione beads bearing plain
GST, GST-K1, or GST-K7 fusion proteins were mixed with purified nuclear
K protein/K protein-associated kinase(s) as in Fig. 6. After
binding, the beads were spun down, washed, divided into 2 aliquots, and
resuspended in phosphorylation buffer while the supernatants were mixed
with 30 µl of glutathione beads containing the full-length GST-K
fusion protein. After binding, GST-K protein beads were washed as
before. Beads from the first (Beads) (left panel, lanes 1-6) and second (Supernatants) (right
panel, lanes 1-6) round of binding were
phosphorylated with (+) or without(-) poly(C) RNA as before (Fig. 6). After phosphorylation, proteins were eluted from the
beads by boiling, separated by SDS-PAGE, stained with Coomassie Blue (upper panels, Coomassie) and autoradiographed (lower
panels,P). Molecular mass standards are
shown in kDa. The diagram shown below the gels illustrates the GST-K
full-length and K deletion fusion proteins used in this
experiment.
Figure 8:
The IL-1-responsive kinase binds and
phosphorylates the COOH terminus fragment of the K protein. EL-4 murine
thymoma cells at 10 cells/ml RPMI 1640 medium were treated
with 10
M IL-1
. At given time points,
5
10
cells were harvested and nuclear extracts were
prepared(12) . A, for each IL-1
time point, 20
µl of protein A beads bearing preimmune rabbit serum were mixed for
2 h (4 °C) with 100 g of nuclear extracts diluted with 1 ml of
binding buffer containing the full complement of phosphatase
inhibitors. After preclearing with the preimmune serum, the beads were
pelleted and the supernatant was mixed with 20 µl of protein A
beads bearing K protein antibody No. 54 (17) (2 h, 4 °C).
Following incubation, beads were washed once with 1.0 ml of binding
buffer containing 175 mM NaCl plus the full complement of
phosphatase inhibitors and twice with 1 ml of binding buffer without
phosphatase inhibitors. Beads were resuspended in 1 ml of binding
buffer containing 4 µM microcystine, and 50 µl of
glutathione beads bearing the GST-K7 mutant (aa 318-464) were
added to each sample. Bead suspension was mixed overnight (4 °C)
and divided into two equal aliquots, and the phosphorylation reaction
was carried out (30 min at 30 °C) as before (Fig. 6) with (lanes 7-11) or without (lanes 1-5) 1
mg/ml poly(C) RNA. The reaction was stopped by washing the beads twice
with binding buffer.
P-Labeled proteins were eluted from
the beads by boiling and were analyzed by SDS-PAGE and autoradiography.
Molecular mass standards are shown in kDa. B, for each
IL-1
time point, 50 µl of glutathione beads bearing both
GST-K13 (aa 1-337) and GST-K1 (aa 425-464) proteins were
mixed overnight (4 °C) with 100 µg of nuclear extracts diluted
with 1 ml of binding buffer containing the full complement of
phosphatase inhibitors. After pelleting, the supernatant was removed
and mixed for 2 h (4 °C) with 50 µl of plain glutathione beads,
and the precleared supernatant was then added to 50 µl of
glutathione beads bearing the GST-K7 mutant. Pelleted beads were then
processed as in A and phosphorylated in the presence (lanes 7-11) or absence (lanes 1-5) of 1
mg/ml poly(C) RNA.
P-Labeled proteins were then analyzed
by SDS-PAGE and autoradiography.
In the above type of an experiment (Fig. 8A) one cannot rule out a possibility that during the phosphorylation reaction the IL-1-responsive KPK merely comes off the immunoprecipitated native K protein, then stays in solution and phosphorylates the GST-K7 fusion protein without meaningful binding to it. To demonstrate, as it was done in Fig. 6, that the GST-K7 deletion mutant actually binds the IL-1-responsive KPK, crude nuclear extracts, rather than immunoprecipitates (Fig. 8A), from the same IL-1 time course were applied to glutathione beads bearing the GST-K7 fusion protein (Fig. 8B). To ensure that the kinase binding is specific for the KPK-BD (aa 318-425), nuclear extracts, which may contain other K protein partners, were first precleared by overnight mixing with glutathione beads containing excess amounts of a mixture made of GST-K13 (aa 1-337) and GST-K1 (aa 425-464) fusion proteins. Supernatants from these beads were then reapplied to plain glutathione beads to clear residual GST-K13 and GST-K1 fusion proteins and, in turn, this supernatant was applied to beads bearing the GST-K7 deletion mutant. After several hours of incubation, the GST-K7 beads were phosphorylated with or without poly(C) RNA as before. An autoradiograph from this experiment shows an IL-1-responsive, poly(C) RNA-facilitated, GST-K7 phosphorylation that was nearly identical with the pattern obtained using K protein/KPK immunoprecipitates (Fig. 8, compare A to B). These two experiments demonstrate that the KPK-BD mapped to the amino acids 318-425 ( Fig. 6and Fig. 7) recruits a kinase that is indeed IL-1-responsive, and that phosphorylation of K protein by this enzyme is facilitated by poly(C) RNA. Because the GST-K13 mutant (aa 1-337) does not retain the KPK (Fig. 8B), we can further narrow down the KPK-binding domain to the amino acids 337-425. KPK may bind to the K protein directly, or the binding may be mediated by another K protein molecular partner. This issue cannot be unequivocally resolved until KPK is cloned and recombinant protein becomes available.
Figure 9:
K protein and KPK co-immunoprecipitate
with c-Src and Vav from EL-4 cell extracts. A, 50 µg of
cytoplasmic protein extracts from EL-4 cells were incubated with 0.5
µl of either antipp60monoclonal antibody (lanes 1 and 2), anti-Vav
C-14 polyclonal antibody (lanes 3 and 4), or anti-K
protein serum No. 54 (17) (lanes 5 and 6)
with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) 750 µM P3
synthetic peptide (see Fig. 5). The immunocomplexes were
recovered with 20 µl of protein A beads (Pharmacia, Uppsala,
Sweden) and centrifugation (IP). Beads were washed once with 1
ml of binding buffer, twice with 1 ml of binding buffer containing 175
mM NaCl, and one more time with 1 ml of binding buffer. After
the washes, beads were boiled in loading buffer, and eluted proteins
were resolved on a 10% SDS-PAGE, electrotransferred to PVDF membranes,
and immunostained (IS) with the anti-K antibody No. 54
(1:10,000 dilution). B, immunoprecipitation, transfer to the
GST-K7 deletion mutant, and phosphorylation were done as illustrated in Fig. 8A. Briefly, 50 µg of cytoplasmic protein
extracts from IL-1-treated EL-4 cells were precleared with protein A
beads bearing preimmune IgG (IP
), and then equal
aliquots of the supernatants were mixed with either anti-K protein
serum No. 54 (lanes 1 and 2), anti-Vav C-14
polyclonal antibody (lanes 3 and 4), or
anti-pp60
monoclonal antibody (lanes 5 and 6). The immunocomplexes were recovered
with 20 µl of protein A beads and centrifugation (IP
). Beads were washed once with 1 ml of binding
buffer, twice with 1 ml of binding buffer containing 175 mM NaCl, and one more time with 1 ml of binding buffer. After
washing, protein A beads were resuspended in binding buffer to which 25
µl of glutathione beads bearing the GST-K7 fusion protein were
added. The beads suspension was mixed for 8 h, then divided into two
equal aliquots, and the phosphorylation reaction was carried out as
before (Fig. 8) with or without 1 mg/ml poly(C) RNA. The
reaction was stopped by washing the beads twice with binding buffer.
P-Labeled proteins were eluted from the beads by boiling
and were analyzed by SDS-PAGE and autoradiography. Molecular mass
standards are shown in kDa.
Because the sites that engage KPK
and the SH3 domains do not overlap, K protein could simultaneously bind
KPK and c-Src or Vav. If so, K protein would serve to promote
multienzyme complex formation. To test whether KPK exists in a complex
with either c-Src or Vav in vivo, c-Src, Vav, and K
immunoprecipitates were prepared from EL-4 cell cytoplasmic extracts
that were first precleared with an excess of protein A beads bearing
preimmune IgG. As before (Fig. 8A), following an
extensive wash, the immunoprecipitates were incubated with a suspension
of beads bearing GST-K7 fusion protein to transfer immunoprecipitated
kinase(s) from the native K protein to the K7 deletion mutant. The
mixed beads were washed several times, and phosphorylation reaction of
the mixed beads was carried out with or without poly(C) RNA (Fig. 9B). Similar to the results with the
immunoprecipitates obtained with the anti-K protein antibody (lanes
1 and 2), the GST-K7 fusion protein bound and was
phosphorylated in a poly(C) RNA-dependent fashion by a kinase that
co-immunoprecipitated with either Vav (lanes 3 and 4)
or c-Src (lanes 5 and 6). These results suggest that
KPK can exist in a complex with c-Src and Vav in vivo, and
that this association is likely mediated by K protein (Fig. 9A). The higher level of GST-K7 phosphorylation
seen with the immunoprecipitates obtained with the anti-K protein
antibody is to be expected since with the anti-K antibody more K
protein was immunoprecipitated compared to the anti-c-Src and anti-Vav
antibodies (Fig. 9A). Also, with the anti-K protein
immunoprecipitates, not all KPK was transferred to the K7 mutant,
accounting for the P-labeled band that represents the
RNA-dependent phosphorylation of the native immunoprecipitated K
protein (Fig. 9B, lane 2).
In order to attribute activation of KPK
to the recombinant c-Src, we carried out the following experiment (Fig. 10) taking advantage of the fact that c-Src can bind to
the GST-K protein SH3-binding domains. The recombinant c-Src
preparation was mixed with glutathione beads bearing either GST-K7
protein or beads bearing a mixture of GST-K13/GST-K1 deletion mutants.
(The GST-K13 mutant contains all three Src SH3-binding sites while
GST-K7 contains none (Fig. 1)). Concurrently, inactivated KPK (17) was co-immunoprecipitated with K protein from crude
nuclear extracts using protein A beads as described above ( Fig. 8and Fig. 9). The glutathione GST-K7 and GST-K13/K1
beads were then spun down, and the respective supernatants were
incubated with K protein/KPK immunoprecipitates to allow binding of
c-Src. After washing extensively, a phosphorylation reaction was
performed on protein A beads in c-Src kinase buffer with or without
poly(C) RNA. An autoradiograph of the P-labeled proteins
eluted from the protein A beads is illustrated in Fig. 10. The
results demonstrated a stronger poly(C) RNA-dependent phosphorylation
of the immunoprecipitated K protein when protein A beads had been
incubated with the supernatant from the GST-K7 fusion protein (lanes 1 and 2) compared to the supernatant from
GST-K13/GST-K1 beads (lanes 3 and 4). Because the
GST-K13 fusion protein depleted c-Src from the supernatant while the
GST-K7 mutant did not, together with the fact that c-Src does not
phosphorylate GST-K protein in a poly(C) RNA-dependent manner, these
results demonstrate that within the context of immunoprecipitated K
protein-KPK complex, c-Src can reactivate KPK poly(C) RNA-dependent
kinase activity. Therefore, these results confirm that the K protein
can simultaneously engage the full-length c-Src and KPK (Fig. 9)
and allow a cross-talk between two K protein partners. Fortuitously, a
very small fraction of the GST-K7 fusion protein (which represents the
KPK-BD, see Fig. 6) was carried over in the supernatant and was
likewise phosphorylated in a poly(C) RNA-dependent fashion (lanes 1 and 2). Because the insect-derived c-Src preparation does
not phosphorylate the GST-K7 protein, the RNA-dependent phosphorylation
of GST-K7 fusion seen here reflects the activity of KPK that was
reactivated by the recombinant c-Src. Collectively, these (Fig. 10) and previous experiments (17) are consistent
with the notion that the serine/threonine KPK engaged by the K protein
can be activated through phosphorylation mediated by a tyrosine kinase
that is recruited to the K protein by the contiguous cluster of
SH3-binding sites, SH3-BD (Fig. 11).
Figure 10:
c-Src stimulates KPK RNA-dependent
phosphorylation of K protein. A, 50 µg of nuclear extracts
were diluted 50-fold with 1 binding buffer containing no
phosphatase inhibitors and incubated for 4 h at 25 °C. Such a
treatment inactivates the K protein kinase(17) . After
inactivation, the phosphatase inhibitor concentration was restored and
the K protein was immunoprecipitated as in Fig. 8. In parallel,
recombinant c-Src preparation (10 units) was precleared for 3 h (4
°C) by mixing with 100 µl of glutathione beads bearing either
GST-K7 (aa 318-464) or beads bearing a mixture of GST-K13 (aa
1-337) and GST-K1 (aa 425-464) deletion mutants resuspended
in 500 µl of 1
binding buffer. After preclearing, the
glutathione beads were spun down and the supernatants from the GST-K7
beads (B, lanes 1 and 2, K7) and from the
GST-K13/GST-K1 beads (B, lanes 3 and 4, K13-K1) were
mixed with protein A beads-immunoprecipitates for 2 h (4 °C). The
immunoprecipitates were then washed once with 1
binding buffer
containing 175 mM NaCl, twice with 1
binding buffer,
and once with Src kinase buffer (100 mM Tris-HCl, pH 7.0, 0.4
mM EGTA, 0.4 mM NaVO
, 40 mM
MgCl
). Phosphorylation was carried out at 30 °C for 15
min in the presence (poly(C) (+)) or absence (poly(C)(-)) of 1 mg/ml poly(C) RNA in c-Src kinase
buffer. Beads were washed twice with 1
binding buffer, and
P-labeled proteins were eluted by boiling. B,
SDS-PAGE and autoradiography of proteins eluted from the beads. Natural
K protein (K) and GST-K7 (GST-K7)
P-labeled bands are shown by arrows. Molecular
mass sizes are shown in kDa.
Figure 11:
Hypothetical model illustrating
interactions between K protein and protein kinases. The K protein is
comprised of modular domains that include three evolutionarily
conserved KH repeats (open rectangles)(46) ,
NH terminus acidic domain, a cluster of three SH3-binding
sites (SH3-BD), and a domain that recruits the IL-1-responsive
K protein kinase (KPK-BD). The results from the studies
presented in this report are consistent with the notion that the K
protein can simultaneously recruit kinases to contiguous domains thus
facilitating cross-talk. One example of such an interaction is
protein-tyrosine kinase (PTK)-mediated phosphorylation of the
K protein kinase which is then activated and, in turn, phosphorylates
the K protein.
In this study we demonstrate that the K protein contains SH3-binding sites. This is in agreement with previous reports that demonstrated in vitro interaction of K protein with the protein-tyrosine kinases Src, Fyn, and Lyn (10, 11) SH3 domains and in vitro and in vivo SH3 interaction of K protein with the proto-oncogene Vav(13, 14) . In the present study we have extended these observations and showed that the K protein, in fact, contains a cluster of at least three SH3-binding domains (Fig. 3Fig. 4Fig. 5), and that in vivo these domains mediate association of K protein with both Vav and c-Src (Fig. 9).
Predictably, we found that the two already described proline-rich sequences, MSPRRGPPPPPPGRG (aa 283-297) and GSRARNLPLPPPPPPRGG (aa 301-318) do serve as SH3-binding sites. Both of these regions contain consensus sequences for class I (underlined, RXXPXXP, where X stands for any amino acid) and class II (in italics, PXXPXR) types of SH3 ligands(35) . We also identified a third SH3 binding motif, MPPGRGGRPMPPSRR (aa 264-278). This site does contain two proline pairs in the context of arginines (36) and does conform well to the consensus sequence for class II SH3 ligands (shown in italics)(35) . The competition experiments using synthetic peptides containing these motifs indicate that these domains engage c-Src SH3 with affinities that differ by more than an order of magnitude. For example, we estimated that the affinity of the P1 region (aa 264-278) for the c-Src SH3 domain is 25-fold lower than that of the P3 region (aa 301-318 stretch) (Fig. 5). These differences suggest that in vivo the P1 region is less likely to engage c-Src than the other two proline-rich sites. Nonetheless, these results demonstrate that K protein contains several clustered SH3-binding sites which could simultaneously engage several of the same or different SH3 domain-containing proteins. Moreover, because the K protein contains consensus sequences for both class I and II types of sites, it could engage SH3 domains in opposite orientations(37) .
Although the linear structure of the most proximal SH3-binding site (aa 264-278) is different from the other two (aa 283-297 and 301-318) proline-rich domains, all three SH3-binding sites exhibited similar selectivity toward Src SH3 and Vav SH3 (Fig. 2Fig. 3Fig. 4). The structural basis for the shared binding specificity of disparate SH3-ligand sequences (29, 37, 38) will have to await three dimensional studies. It is interesting to note that all three K proline-rich regions have an adjacent RGG motif, a site that is methylated by a specific methyltransferase(39) . It has been suggested that methylation of these residues might regulate SH3 interactions(40) . Because K protein is arginine-methylated in vivo(39) , this modification may be one way by which the interaction of K protein with its many molecular partners is regulated.
Each of the three SH3-binding domains can independently and specifically engage c-Src and Vav SH3 domains (Fig. 2Fig. 3Fig. 4Fig. 5). These domains are contiguous with a domain that recruits an IL-1-responsive kinase that phosphorylates K protein in an poly(C) RNA-dependent manner (Fig. 6Fig. 7Fig. 8). The functional significance of the succession of several SH3-ligand sites followed by a kinase binding domain may reflect the ability of the K protein to bind simultaneously two or more proteins that contain SH3 domains, for example c-Src and Vav, in addition to other enzymes that bind to the adjacent domains. As such, the K protein may serve as a docking platform for multiple enzymes. Such a model is supported by the ability of the K protein to simultaneously engage in vivo KPK and c-Src or Vav (Fig. 9). Moreover, the ability of c-Src, once recruited to the K protein, to activate K protein-bound KPK (Fig. 10), indicates that the K protein not only provides docking sites but in fact may facilitate intermolecular communication (Fig. 11). As such, the function of K protein would be analogous to the role of the insulin receptor substrate (IRS-1) which recruits p85, Grb-2, and the protein-tyrosine phosphatase, Syp(41) , and to the yeast Ste5 which facilitates interactions for kinases in the MAP kinase cascade(42, 43) . Notably, in the case of the K protein, phosphorylation of the COOH terminus region is modulated by RNA (Fig. 6Fig. 7Fig. 8); therefore, the cross-talk among the multiple proteins that are docked on the K protein might be regulated by cognate nucleic acid motifs.
The molecular mechanisms responsible for the IL-1-responsive phosphorylation of K protein (12, 17) remain to be defined. However, given the observation that KPK appears to be activated by phosphorylation(17) , recruitment of c-Src or a related tyrosine kinase to K protein may serve to facilitate phosphorylation and thus activation of KPK (Fig. 10Fig. 11). Given the fact that IL-1 does activate protein-tyrosine kinases(33, 34) , recruitment of this class of kinase to the SH3-binding domains may provide a mechanism by which KPK(17) , which is engaged by a contiguous domain, is activated in response to IL-1 (Fig. 11).
In summary, this study shows that the K protein contains a series of SH3-binding sites that is contiguous with a domain that binds an IL-1-responsive kinase. K protein can simultaneously engage proteins and nucleic acids (Fig. 8) and appears to facilitate molecular interactions ( Fig. 10and Fig. 11). Based on these observations, K protein may belong to a new class of molecules which act as docking proteins for molecules involved both in signal transduction and gene expression. This class of proteins may in addition include p62/p68 or Sam68, which has also been postulated to function as a docking protein(40) . Like K protein, Sam68 contains SH3-binding and KH domains, is a target for c-Src(11, 15) , has multiple molecular partners, and binds RNA and DNA (11, 15, 40, 44) .