(Received for publication, July 31, 1996, and in revised form, November 14, 1996)
From the Polypeptide Laboratory, Division of Endocrinology, Department of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada
Nck is a 47-kDa cytosolic protein devoid of
intrinsic catalytic activity and consisting of Src homology 2 and 3 (SH2 and SH3) domains organized as follows: SH3-SH3-SH3-SH2. Nck is
believed to act as an adaptor protein mediating signal transduction
initiated by receptor tyrosine kinases (RTKs). Through its SH2 domain,
Nck recognizes a specific phosphotyrosine residue on RTKs or on protein substrates of RTKs like insulin receptor substrate-1, the major substrate of the insulin receptor, and through its SH3 domains it
interacts with poorly characterized effector molecules. To identify
novel proteins that might interact with Nck, we have used the
amino-terminal segment of Nck encompassing its three SH3 domains in the
yeast two-hybrid system. Among the polypeptides that associate with
Nck, we have identified the 2 isoform of the serine/threonine casein
kinase I (CKI-
2). In transformed rat hepatocytes overexpressing the
insulin receptor (HTC-IR cells), serine/threonine protein kinase
activity coimmunoprecipitates with Nck, an interaction mediated mainly
by the third SH3 domain of Nck. This kinase activity is not apparently
modulated by insulin, nor is it sensitive to staurosporine or heparin,
and it does not use GTP as a phosphate donor. However the kinase
activity coimmunoprecipitated with Nck is completely abolished by
N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a
specific inhibitor of casein kinase I. In an in vitro
renaturation gel kinase assay, a protein kinase of 70-75 kDa was
detected associated with the SH3 domains of Nck. Far Western analysis
demonstrated that the SH3 domains of Nck bound directly to a cytosolic
protein of 70-75 kDa. A rabbit polyclonal antibody raised against the C-terminal region of CKI-
2 protein kinase immunoprecipitated a
single specific protein of 70-75 kDa from HTC-IR cell lysates and
detected CKI-
2 among the proteins coimmunoprecipitated with Nck.
These results support an in vivo interaction between Nck and CKI-
2 and suggest that CKI-
2 could be involved in signaling pathways downstream of RTKs.
Common events triggered by membrane receptor tyrosine kinases (RTKs)1 involve activation of the intrinsic receptor tyrosine kinase activity, autophosphorylation, and tyrosine phosphorylation of various intracellular proteins (1). Phosphorylation of specific tyrosine residues creates high affinity binding sites for a variety of cytoplasmic SH2 domain-containing proteins (2), which are recruited to propagate signals to downstream effector molecules (3). SH2 domains mediate protein-protein interactions by direct recognition of phosphotyrosine-containing motifs (4, 5), and their role in the assembly of protein complexes has been firmly established (6). Many of the SH2 domain-containing proteins possess one or several SH3 domains, which mediate protein interactions through the recognition of proline-rich sequences in their target molecules (7, 8). The importance of SH3 domain-mediated interactions was first revealed with Grb2, an adaptor molecule composed almost exclusively of SH2 and SH3 domains. Grb2 has been implicated in a highly conserved mechanism for the control of the Ras-dependent activation of mitogen-activated protein kinase by RTKs. Mutations in the SH3 domains of Sem5, the Caenorhabditis elegans homologue of Grb2 (9), have been reported to impair its ability to transmit a biological signal (10). Interestingly, the finding that an SH3 domain and a proline-rich sequence also mediate a direct interaction between two components of the phagocyte NADPH oxidase complex (11), suggests that the role of SH3-mediated protein interactions could be expanded to regulatory molecules involved in cell responses unrelated to cell division. These observations stress the importance of identifying proteins that bind SH3 domains.
Nck is a widely expressed 47-kDa cytoplasmic protein (12) consisting of SH2 and SH3 domains organized in the order SH3-SH3-SH3-SH2 (13). Nck has been shown to bind via its SH2 domain to the platelet-derived growth factor and epidermal growth factor receptors (12, 14, 15) and intracellular tyrosine-phosphorylated proteins, such as IRS-1 (16), and c-Src (15). Activation of growth factor RTKs induces Nck phosphorylation on tyrosine and serine residues (12, 14, 15, 17). In fibroblasts, overexpression of Nck leads to cell transformation (12, 15). Despite these suggestions of its involvement in mitogenesis and oncogenesis, studies examining the role of Nck in mediating these processes are sparse. By analogy to the crucial role played by Sos, which associates with the Grb2 SH3 domains, in the activation of Ras-dependent signaling (18, 19), effector proteins interacting with the SH3 domains of Nck may also trigger cellular signaling events leading to biological responses. In fact, Nck, through its SH3 domains, has been shown to interact with effectors involved together with small GTPases in the regulation of the cytoskeleton (20), which include Sos (21), an activator of Ras, and mPAK3 (22), a serine/threonine kinase activated by Rac1 and Cdc42, members of the Rho-GTPase family. Interestingly, Dock, a Drosophila adaptor protein homologous to human Nck, has been reported to be required for normal photoreceptor R cell axon guidance and targeting (23), providing further support for Nck involvement in regulating cytoskeletal organization. In addition, c-Cbl (24), Nck-associated kinase (NAK; a serine/threonine kinase) (25), and the Wiskott-Aldrich syndrome protein (WASP) (26) also interact with the SH3 domains of Nck; however, the functional significance of these interactions is currently unknown.
To identify novel proteins that might interact with Nck, we have used
the amino-terminal domain of Nck encompassing its three SH3 domains, in
the yeast two-hybrid system (27). Among the polypeptides we found
associating Nck, was a protein very similar to the 2 isoform of the
rat serine/threonine casein kinase I (CKI-
2) (28). In the present
study, we have examined the relation between Nck and CKI-
2 in an
insulin-responsive cell line and found that in transformed rat
hepatocytes overexpressing the insulin receptor, CKI-
2 was
constitutively associated with Nck.
Transformed rat hepatocytes overexpressing the human insulin receptor (HTC-IR cells) (30) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal calf serum (Life Technologies) and maintained in selection medium containing 40 µg/ml of Geneticin (G418) (Life Technologies). Subconfluent HTC-IR cells were serum-starved in Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin (Sigma), 24-48 h prior to the experiment. In experiments involving insulin stimulation (porcine insulin, Connaught-Novo Laboratories, Willowdale, Canada), insulin was added to cells in culture (final concentration 100 nM) for the times indicated in the figure legends.
AntibodiesA polyclonal Nck antibody was raised in rabbits,
by immunizing with a GST fusion protein containing the SH3 domains of
human Nck (residues 1-251). A polyclonal CKI-2 antibody was
produced by immunizing rabbits with a GST fusion protein containing the C-terminal portion of the CKI-
2 (residues 192-414). This was obtained by subcloning the partial CKI-
2 cDNA isolated from the two-hybrid screen into pGEX4T2 (Pharmacia Biotech Inc.).
HTC-IR
cells were lysed in the following buffer: 50 mM HEPES, pH
7.5, 150 mM sodium chloride, 10% (v/v) Triton X-100, 1.5 mM magnesium chloride, 1 mM EGTA, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride, 10 mM sodium fluoride, 10 mM sodium
pyrophosphate, 200 µM sodium orthovanadate, and 40 mM -glycerophosphate. Clarified lysates were adjusted
with lysis buffer to contain a final protein concentration of 1 mg/ml
and were then submitted to Nck immunoprecipitation at 4 °C, using our polyclonal rabbit antibody (see above) and protein A-Sepharose (Sigma). After 90 min of gentle agitation, the protein
A-Sepharose beads were washed three times with lysis buffer and once
with kinase reaction buffer (20 mM HEPES, pH 7.5, 1 mM dithiothreitol, 5 mM MgCl2, 10 mM
-glycerophosphate) and were resuspended in 25 µl of
kinase buffer. In experiments where kinase activity was assayed on
exogenous substrates, 15 µg of dephosphorylated
-casein (Sigma), 5 µg of myelin basic protein
(Sigma), 15 µg of
-casein (Sigma), 5 µg of histone HI
(Sigma), 15 µg of phosvitin
(Sigma), or 15 µg of PolyGluTyr
(Sigma) was added to the assay. After preincubation at
30 °C for 5 min, the phosphorylation assay was initiated by adding
[
-P32]ATP (20 µM, 10 µCi) (DuPont
NEN), and after 20 min at 30 °C, the reaction was stopped by the
addition of 6 µl of 6 × Laemmli buffer (31) followed by boiling
for 2 min before subjecting to SDS-PAGE. Phosphorylation of exogenous
substrates or proteins coimmunoprecipitated with Nck were
analyzed following SDS-PAGE and autoradiography.
Guanosine triphosphate (100 µM) (Sigma), heparin (200 µg/ml)
(Sigma),
N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7)
(25-100 µM) (Seikagaku Corp.), or staurosporine (10 µM) (Sigma) were added to the kinase
reaction assay before the 5-min preincubation period at 30 °C. The
reactions were then initiated by the addition of
[P32]ATP as reported above. Phosphorylation of
-casein or proteins coimmunoprecipitated with Nck was analyzed
following SDS-PAGE and autoradiography.
Phosphoamino acid analysis of
-casein band was performed following SDS-PAGE, transfer of the
proteins to Immobilon-P, and excision of the radioactive
-casein
band. The latter was incubated in 6 N HCl at 110 °C for
1 h. The hydrolysates were separated by two-dimensional
electrophoresis (32). The 32P-labeled phosphoamino acids
were detected by autoradiography and compared with ninhydrin-stained
phosphoamino acid standards (Sigma).
GST fusion proteins containing various domains of wild type human Nck (GST-SH2, residues 282-377; GST-SH3, residues 1-251; GST-Nck, full-length cDNA) were produced by polymerase chain reaction using specific oligonucleotide primers containing the appropriate restriction sites, subcloned into pGEX4T2 or pGEX2TK plasmids (Pharmacia) and sequenced. For SH3 mutants, a point mutation was performed in individual SH3 domains by extension overlapping polymerase chain reaction (33). In each SH3 domain, the first tryptophan residue (Trp38, Trp143, Trp229) of the tryptophan doublet, well conserved among several SH3 domains, was mutated to arginine. The mutations were confirmed by dideoxynucleotide sequencing (Pharmacia), and the proteins were expressed and immobilized on glutathione-agarose beads. Equal amounts of each fusion protein (2 µg) were incubated with HTC-IR cell lysates for 90 min at 4 °C, after which proteins bound to GST fusion proteins immobilized on beads were assayed for associated kinase(s).
In Vitro Renaturation Kinase Assay-Casein (50 µg/ml)
was dissolved in the polyacrylamide gel solution just prior to
polymerization. Protein kinases from HTC-IR cell lysates interacting
with the GST fusion proteins of various domains of Nck or
coimmunoprecipitating with Nck were detected directly in the gel, by
their ability to phosphorylate the
-casein substrate in the gel
(34). Samples were subjected to SDS-PAGE, and SDS was removed by
washing with 20% (v/v) isopropyl alcohol in 50 mM
imidazole, 28 mM iminodiacetic acid, pH 8.0, twice for 60 min at room temperature. The gels were then washed with 50 mM imidazole, 28 mM iminodiacetic acid, pH 8.0, containing 10 mM mercaptoethanol for 60 min at room
temperature. Proteins in the gels were then denaturated with 8.0 M guanidine HCl in 50 mM imidazole, 25 mM iminodiacetic acid, pH 8.0, containing 50 mM
mercaptoethanol for 90 min at room temperature. Protein renaturation was achieved by successive washes at 4 °C (2 × 90 min, 200 ml; overnight, 400 ml; 1 × 60 min, 200 ml) in 25 mM
imidazole, 14 mM iminodiacetic acid, pH 8.0, containing 20 mM KCl, 10% sucrose, 10 mM mercaptoethanol,
1% bovine serum albumin, and 0.04% Tween 20. The gels were
equilibrated in a solution containing 10 mM HEPES (pH 8.0),
10 mM mercaptoethanol, 5 mM MgCl2,
and 10 mM
-glycerophosphate for 60 min at room
temperature and incubated for an additional 120 min following the
addition of [
-32P]ATP (20 µCi/ml). Finally, the gels
were extensively washed with 5.0% (w/v) trichloroacetic acid
containing 1.0% (w/v) sodium pyrophosphate and 1.0% sodium phosphate.
After fixing and drying, the gels were exposed for autoradiography.
Total cell lysates (20 µg of proteins) from control or insulin-stimulated HTC-IR cells were resolved by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked in 20 mM HEPES, pH 7.5, 5 mM MgCl2, 1 mM KCl, 5 mM dithiothreitol, 5 mM NaF, 0.02% sodium azide, 5% nonfat dry milk for 24 h at 4 °C and probed overnight at room temperature with 32P-labeled GST or 32P-labeled GST-SH3 Nck (specific activity 1 × 106 cpm/ml in blocking buffer) prepared according to the manufacturer's recommendations (pGEX2TK; Pharmacia). At the end of the incubation period, the membranes were extensively washed in Tris-buffered saline containing 0.1% Triton X-100 and exposed for autoradiography.
Nck and CKI-HTC-IR cells were
harvested in buffer A (5 mM Tris-HCl, pH 7.4, 1 mM benzamidine, 1 mM phenylmethylsulfonyl
fluoride, 2 mM NaF, 2 mM
Na3VO4, 1 mM MgCl2, and
0.25 M sucrose and homogenized using a Teflon-glass
homogenizer. After centrifugation at 15,000 rpm for 10 min, the
supernatant was gently agitated with anti-Nck or anti-CKI-2 rabbit
polyclonal antibodies and protein A-Sepharose beads at 4 °C. After
90 min, the beads were washed twice with cold phosphate-buffered
saline, resuspended in Laemmli buffer, boiled for 2 min, and loaded
on a 7.5% acrylamide gel. Proteins were transferred onto
nitrocellulose, and the membrane was blocked in 5% milk. CKI-
2 was
detected using our rabbit polyclonal CKI-
2 antibody, the horseradish
peroxidase-coupled sheep anti-rabbit antibody (Bio-Rad), and the
enhanced chemiluminescence reagent (ECL; Amersham Corp.).
Using the yeast two-hybrid system, we have isolated a
kinase-related cDNA encoding a protein that interacts with the
three SH3 domains of Nck and whose amino acid sequence is identical to
the C-terminal domain of rat CKI-2, which contains a unique proline-rich sequence (28). To verify an interaction between Nck and
CKI-
2 in intact cells, we immunoprecipitated Nck from unstimulated
HTC-IR cell lysates and assayed for in vitro protein kinase
activity using a variety of proteins as exogenous substrates. As shown
in Fig. 1, a protein kinase activity was
coimmunoprecipitated with Nck and preferentially phosphorylated myelin
basic protein,
-casein, histone H1, and phosvitin as compared to
-casein and PolyGluTyr. The kinase activity toward
-casein was
magnesium-dependent (Fig. 2A). When magnesium
and manganese were combined in the same assay, a lesser degree of
kinase activity was observed (data not shown). Phosphoamino acid
analysis of phosphorylated
-casein revealed specific phosphorylation
of serine and threonine residues, demonstrating that the Nck-associated
kinase is a serine/threonine protein kinase (Fig. 2B).
Effect of Insulin and Protein Kinase Inhibitors
We next
studied the effect of treating cells with insulin and various drugs on
in vitro protein phosphorylation in Nck immunoprecipitates (Fig. 3A). Insulin, when added for 5 min to
the cells in culture, did not affect the kinase activity
coimmunoprecipitated with Nck. Staurosporine, an inhibitor of the
protein kinase C family, cAMP- and cGMP-dependent kinases,
Ca2+/calmodulin-dependent protein kinase, and
myosin light chain kinase (35), had no effect in vitro on
the kinase activity, thus excluding these kinases as likely candidates.
To evaluate the presence of casein kinases in Nck immunoprecipitates,
we tested the effect of heparin (a known inhibitor of casein kinase II
(CKII)) (36), GTP (which could be used as a phosphate donor by CKII but
not by casein kinase I (CKI)) (37), and CKI-7 (a specific inhibitor of
CKI activity) (38). Neither heparin nor GTP had an effect on protein
phosphorylation in Nck immunoprecipitates, suggesting that CKII is not
involved. However, CKI-7 strongly inhibited protein phosphorylation in
Nck immunoprecipitates (Fig. 3A) and also prevented in
vitro phosphorylation of -casein (Fig. 3B) with an
IC50 of 20 µM, which is in the range of
concentrations for the inhibitory effect of CKI-7 on purified (38) or
recombinant CKI activity (28, 39). Taken together, these results are
highly suggestive of an interaction between Nck and a member of the CKI
family.
To determine whether the Nck-associated protein kinase activity could
be modulated by insulin over a longer time course, insulin was added to
the cells up to 30 min prior to the preparation of cell lysates. The
effects of insulin on Nck-associated kinase activity were evaluated by
in vitro phosphorylation of the overall proteins
coimmunoprecipitated with Nck or by in vitro phosphorylation of -casein added in the assay. When
-casein was used as an
exogenous substrate, insulin stimulation did not seem to affect the
kinase activity associated with Nck (Fig. 4,
bottom). On the other hand, when the overall in
vitro phosphorylation of proteins coimmunoprecipitated with Nck
was analyzed, some proteins appeared less phosphorylated when the cells
were stimulated 15 and 30 min by insulin (Fig. 4, top),
suggesting that the kinase activity toward specific protein substrates
might be regulated by insulin. These experiments did not discriminate
between a change in the specific activity of the Nck-associated kinase
and the amount of proteins associated with Nck upon insulin
stimulation. A decrease in either could produce an apparent reduced
phosphorylation of target proteins
The Third SH3 Domain of Nck Mediates the Interaction with the Kinase
To determine which domain of Nck mediates the interaction
with the CKI-like protein kinase, GST fusion proteins containing various Nck domains were incubated with HTC-IR cell lysates, and kinases bound to these GST fusion proteins were assayed using -casein as substrate. Fig. 5 demonstrates that the
SH3 domains of Nck associate with protein(s) containing kinase
activity. In fact, the protein(s) with a kinase activity bound equally
well to the GST fusion protein encoding the three SH3 domains of Nck (GST-SH3(3)) as it did to the full-length Nck (GST-Nck), but neither the fusion protein expressing the SH2 domain of Nck nor GST itself could interact with a kinase mediating in vitro
phosphorylation of
-casein. The fusion proteins GST-SH3(3) and
GST-Nck were also phosphorylated in this assay (data not shown),
suggesting that Nck was a substrate of this kinase.
We attempted to identify which of the three SH3 domains of Nck
interacts with the protein kinase by site-directed mutagenesis. Thus,
we created single point mutations in each SH3 domain by substituting
for arginine the first tryptophan residue (Trp38,
Trp143, Trp229) of the well conserved
tryptophan doublet. This particular point mutation of SH3 domains has
been reported to reduce dramatically the ability of SH3 domains to
interact with specific proteins (40, 41). Mutation in the first SH3
domain of Nck had no effect on the kinase activity recruited from
HTC-IR cell lysate; however, mutations of the second and the third SH3
domain were accompanied by a sharp decrease in Nck-associated kinase
activity. This effect was greater with a mutation in the third, rather
than the second SH3 domain (Fig. 6).
Identification of the Nck-associated Protein Kinase
To
identify the protein kinase associated with Nck, an in vitro
renaturation kinase assay in acrylamide gel was performed (Fig.
7A). This procedure allows detection of
protein kinase in the gel by their ability to phosphorylate an
exogenous substrate embedded throughout the gel. GST fusion proteins
encoding the SH3 domains of Nck as well as the whole Nck protein
associated with a protein kinase of 70-75 kDa, which phosphorylates
-casein present in the gel (Fig. 7A). A similar protein,
which phosphorylates
-casein, was also detected in Nck
immunoprecipitates (Fig. 7A). In a direct binding assay (Far
Western) on total cellular proteins immobilized on nitrocellulose, the
32P-labeled GST fusion protein encoding the three SH3
domains of Nck strongly associated with a protein of 70-75 kDa, and
this association was not modulated by prior stimulation of cells with insulin (Fig. 7B). Other protein species of different
molecular mass (55, 120, 190, and 230 kDa) also interacted directly
with the three SH3 domains of Nck; however, in the in vitro
renaturation kinase assay, none possessed kinase activity.
Interestingly, association of the three SH3 domains of Nck with the
120-kDa protein (Fig. 7B) seems to be strongly reduced by
prior cell stimulation with insulin, suggesting that insulin could
modulate association of specific effector proteins with the SH3 domains
of Nck. When 32P-labeled GST was used to probe an identical
membrane, no protein association was detected (data not shown). A
rabbit polyclonal antibody against the C-terminal domain of casein
kinase I-
2 (cDNA obtained from the yeast two-hybrid screen)
recognized, in HTC-IR cell lysates, a specific protein of 70-75 kDa
(inhibited by an excess of antigen; data not shown) (Fig.
7C), which is identical to the molecular mass of the
Nck-associated protein kinase detected in the in vitro
renaturation gel kinase assay and with one of the proteins associated
with the SH3 domains of Nck in the direct binding assay. CKI-
2 was
identified in Nck immunoprecipitates from HTC-IR cells when cytosolic
fractions were prepared in the absence of Triton X-100 (Fig.
7D). When 1% Triton X-100 was added to the cell extract
before the immunoprecipitation of Nck, it reduced the specific
interaction between Nck and CKI-
2 to below the level of protein
detection that could be observed in Western blot analysis of
protein-protein interactions (data not shown).
CKI-2, a serine/threonine protein kinase
of 70-75 kDa is constitutively associated with Nck. A,
in vitro gel kinase assay. Unstimulated HTC-IR cells lysed in 1% Triton lysis buffer were
immunoprecipitated with antibodies to Nck or mixed with GST fusion
proteins containing various domains of Nck. Bound proteins were
fractionated by SDS-PAGE and subjected to an in vitro gel kinase assay as described under "Experimental Procedures."
B, Far Western analysis. HTC-IR cells, unstimulated or
stimulated with 100 nM insulin for 5 min, were lysed in 1%
Triton lysis buffer. Proteins (20 µg) were resolved by SDS-PAGE and
transferred to nitrocellulose. Membranes were blocked 24 h at
4 °C in 20 mM Hepes, pH 7.5, 5 mM
MgCl2, 1 mM KCl, 5 mM
dithiothreitol, 5 mM NaF, 0.02% NaN3, and 5%
nonfat dry milk and then probed overnight at room temperature with
32P-labeled GST-SH3 domains of Nck (106 cpm/ml)
in the same buffer. Membranes were extensively washed in Tris-buffered
saline containing 0.1% Triton and exposed for autoradiography.
C, CKI-
2 antibodies. Unstimulated HTC-IR cell lysate was
immunoprecipitated with antibodies to CKI-
2 (described under
"Experimental Procedures") or normal serum. Immunoprecipitated proteins and proteins from total cell lysate (20 µg) were resolved by
SDS-PAGE and transferred to nitrocellulose. Membranes were blocked, and
CKI-
2 was revealed using antibodies to CKI-
2, protein A-horseradish peroxidase, and ECL. D, coimmunoprecipitation
of Nck-CKI-
2. Unstimulated HTC-IR cells were harvested in 50 mM Tris-HCl, pH 7.4, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 2 mM NaF, 1 mM MgCl2, and 0.25 M sucrose and
homogenized using a Teflon-glass homogenizer. Lysates were
immunoprecipitated with antibodies to Nck, CKI-
2, or normal serum.
Immunoprecipitated proteins were fractionated by SDS-PAGE, transferred
to nitrocellulose, and immunoblotted with antibodies to CKI-
2.
Detection was achieved using goat-anti rabbit-HRP and ECL.
Nck is a substrate of various cell surface tyrosine kinase receptors such as the epidermal growth factor and the platelet-derived growth factor receptors (12, 14, 17). It also appears to be a substrate for a variety of serine/threonine kinases activated by several membrane receptors, like protein kinase C and protein kinase A (cAMP-dependent protein kinase) (12, 14, 17) and has been reported to be highly phosphorylated on serine residues in vivo in resting cells (14). Recently, two independent groups have demonstrated a constitutive physical association between serine/threonine protein kinases and the SH3 domains of Nck, providing a potential explanation for such a high level of serine phosphorylation of Nck in resting cells. First, NAK a 65-kDa serine/threonine kinase, has been reported to interact with the second SH3 domain of Nck (25). However, neither its biochemical characterization nor its exact role in mediating Nck signaling was described. Second, a serine/threonine kinase, mPAK-3 (p21 (Cdc42/Rac1)-activated kinase) was shown to specifically bind to at least one SH3 domain of Nck (22). The kinase activity of mPAK-3, as observed for other members of the PAK family of serine/threonine protein kinases, is increased by activated Rac1 and Cdc42, the small GTP-binding proteins involved in the characteristic cytoskeletal rearrangements associated with cell motility (20, 42). This raises the possibility that Nck is involved in cytoskeletal interactions through its interaction with mPAK-3.
In this study, we have demonstrated that in HTC-IR cells, Nck is constitutively associated with a serine/threonine protein kinase whose activity is magnesium-dependent. This association is mediated particularly by the third SH3 domain, although the second SH3 domain in the linear sequence might be involved in stabilizing the interaction. These results contrast with those for NAK and mPAK3, which have been shown to bind exclusively to the second SH3 domain of Nck (25, 43). This supports our conclusion that in HTC-IR cells the serine/threonine protein kinase coimmunoprecipitated with Nck differs from NAK and mPAK3. In addition, it is possible that NAK and PAK3 could be the same protein kinase, because both are 65 kDa and are found associated to the same SH3 domain of Nck.
Several lines of evidence support the identity of the Nck-associated
kinase in HTC-IR cells as a member of the CKI family of protein
kinases. First, CKI-7 a specific inhibitor for CKI enzymes completely
inhibited the activity of the protein kinase coimmunoprecipitated with
Nck. Second, the Nck-associated kinase could be distinguished from CKII
by its inability to use GTP as a phosphate donor and by its weak
sensitivity to heparin. Furthermore, numerous kinases were ruled out by
the absence of effect of staurosporine. Although staurosporine is a
broad spectrum inhibitor of serine/threonine and tyrosine kinase
activities, it has not been reported as an inhibitor of the casein
kinase family of protein kinases (35). Moreover, the fact that the
kinase activity coimmunoprecipitated with Nck was not (at least
apparently) affected by prior treatment of cells with insulin is in
agreement with the fact that casein kinase family members are second
messenger-independent (37). Finally, we have demonstrated that
CKI-2, a member of the CKI family of protein kinases (28),
coimmunoprecipitates with Nck in vivo. Among casein kinase
I-
isoforms, CKI-
2 is the only one that contains a proline-rich
sequence (28). Moreover, this unique proline-rich sequence in the
C-terminal region of CKI-
2 is composed of two juxtaposed
PXXP motifs
(VH
DV
SQ
PHR), which are
recognized as consensus motifs for SH3-ligand interactions and could
support the constitutive association of CKI-
2 with Nck.
In insulin signal transduction, the importance of IRS-1 has been firmly
established (44-46). Following insulin receptor activation, tyrosine-phosphorylated IRS-1 has been shown to regulate PI-3 kinase
activity by its direct association with p85, the regulatory subunit of
PI-3 kinase. Binding of p85 to IRS-1 leads to activation of the p110
catalytic domain of PI-3 kinase (47, 48). Similarly, IRS-1 has been
reported to associate with Grb2 upon insulin stimulation (49). The
binding of Grb2 to IRS-1 results in recruitment of Sos to IRS-1, thus
providing a link between the activation of the Ras signaling pathway
and insulin receptor activation (50). Nck is found associated with
IRS-1 upon insulin stimulation (16), suggesting that this complex could
regulate specific effector molecules bound to Nck. Unlike Grb2 and p85,
the associated catalytic components of Nck are not yet well
characterized, and its biological function in insulin signaling remains
uncertain. Our finding that Nck is constitutively bound to CKI-2 in
HTC-IR cells could implicate CKI-
2 in insulin signaling. It is
possible that following insulin receptor activation, the binding of Nck
to IRS-1 may regulate the catalytic activity of CKI-
2 or result in
its translocation to specific subcellular sites, thus bringing it in
proximity to substrate molecules. Interestingly, the
-subunit of the
insulin receptor has been reported to be phosphorylated by casein
kinase I in vitro (29), suggesting that if this happens
in vivo, casein kinase I might participate in regulating
insulin receptor activity. Alternatively, IRS-1 could be a target of
casein kinase I, since several consensus motifs for phosphorylation by
casein kinase I (XEXXS*X, where
X is any residue, E is glutamic acid, and S* is the
phosphoserine) (51) are present in the amino acid sequence of IRS-1. Of
course, the presence of consensus motifs for phosphorylation does not
ensure that IRS-1 is phosphorylated by CKI-
2 in vivo. However, the serine/threonine phosphorylation of IRS-1 by an
unidentified kinase was reported to abrogate insulin-stimulated glucose
transport (52), suggesting a mechanism of negative feedback regulation triggered by activation or translocation of serine/threonine kinases following insulin stimulation. Casein kinase I reported to be associated to the p75 tumor necrosis factor receptor, plays an important role in the negative regulation of tumor necrosis factor receptor signaling (53). Hypothetically, CKI, by phosphorylating the
insulin receptor and/or IRS-1, could exert a similar effect on
insulin-activated signaling pathways.
In insulin-stimulated cells, since Nck forms a stable complex with IRS-1 (16), it is suggested that the insulin receptor could regulate the function of Nck and consequently the activity of effector molecules associated with the SH3 domains of Nck. The level of Nck was increased in epididymal fat and in liver from obese insulin-resistant diabetic mice (KKAy) (54) and significantly decreased in liver of streptozotocin-treated rats (55). These findings suggest that changes in the level of Nck expression might contribute to altered insulin responsiveness involving modification of the Nck downstream signaling components.
Insulin is well known to induce increases in the rates of RNA and
protein synthesis in many tissues (56). These effects require the
insulin receptor and IRS-1 (57). More specifically, insulin appears to
regulate the serine/threonine phosphorylation of several elongation and
initiation factors involved in controlling protein synthesis (58-60),
and in vitro studies have demonstrated that these factors
are phosphorylated by several kinases, including CKI (61-63). Thus,
CKI could be required for insulin regulation of protein synthesis. The
finding that Nck associates with CKI-2 demonstrates a new mechanism
by which the insulin receptor can couple to unidentified signaling
molecules. Further work is needed to characterize the interaction
between Nck and CKI-
2 and to determine the downstream elements
regulated by this association.
We thank Christian Band for critical review of this manuscript.