Departments of Medicine and Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0711
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ABSTRACT |
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Endogenous IGF-I regulates growth of human intestinal smooth muscle cells by jointly activating phosphatidylinositol 3-kinase (PI3K) and ERK1/2. The 70-kDa ribosomal S6 kinase (p70S6 kinase) is a key regulator of cell growth activated by several independently regulated kinases. The present study characterized the role of p70S6 kinase in IGF-I-induced growth of human intestinal smooth muscle cells and identified the mechanisms of p70S6 kinase activation. IGF-I-induced growth elicited via either the PI3K or ERK1/2 pathway required activation of p70S6 kinase. IGF-I elicited concentration-dependent activation of PI3K, 3-phosphoinositide-dependent kinase-1 (PDK-1), and p70S6 kinase that was sequential and followed similar time courses. IGF-I caused time-dependent and concentration-dependent phosphorylation of p70S6 kinase on Thr421/Ser424, Thr389, and Thr229 that paralleled p70S6 kinase activation. p70S6 kinase(Thr421/Ser424) phosphorylation was PI3K dependent and PDK-1 independent, whereas p70S6 kinase(Thr389) and p70S6 kinase(Thr229) phosphorylation and p70S6 kinase activation were PI3K dependent and PDK-1 dependent. IGF-I elicited sequential Akt(Ser308), Akt(Ser473), and mammalian target of rapamycin(Ser2448) phosphorylation; however, transfection of muscle cells with kinase-inactive Akt1(K179M) showed that these events were not required for IGF-I to activate p70S6 kinase and stimulate proliferation of human intestinal muscle cells.
insulin-like growth factor-I; proliferation; protein kinase C; protein kinase B; Akt; extracellular signal-regulated kinase 1/2; and p38 mitogen-activated protein kinase
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INTRODUCTION |
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IGF-I REGULATES
CELLULAR proliferation in two ways: 1) it is mitogenic
for many cells and 2) it promotes the increase in cell size
that is required for cell division. Binding of IGF-I to the cognate
IGF-I receptor stimulates the intrinsic tyrosine kinase activity of the
receptor. Mutational analysis has identified several tyrosine
phosphorylation sites within the carboxy terminus of the IGF-I receptor
-subunit that confer specificity for the proliferative effects
elicited in response to receptor autophosphorylation: Tyr950, Tyr1131, Tyr1135, and
Tyr1136 (20, 28, 29). The activated IGF-I
receptor tyrosine kinase has several known primary substrates,
including the insulin receptor substrate family of proteins,
Src-homology domain carboxy terminus, Grb2-associated binder 1, hepatocyte plasma membrane ecto ATPase (pp120/HA4), and G2 kDa
GAP-associated docking protein (p62DOK). Once phosphorylated,
these docking proteins activate downstream intracellular signaling
through the phosphatidylinositol 3-kinase (PI3K) or Grb2-SOS pathways
that ultimately leads to cellular proliferation. The specific pathways
activated by IGF-I and the roles of these pathways in mediating the
proliferative response to IGF-I depend on the cell type. We have
previously shown that in human intestinal smooth muscle cells, like
skeletal muscle cells and vascular smooth muscle cells, IGF-I activates
both the ERK1/2 and PI3K pathways that regulate growth (11, 14,
24). The particular importance of IGF-I in regulating the growth
of intestinal smooth muscle is demonstrated by the hyperplasia that occurs in both intestinal and vascular smooth muscle tissues of transgenic animals overexpressing an IGF-I cDNA (31, 48). The pathways mediating intestinal smooth muscle proliferation are
likely to play an important role in the setting of Crohn's disease, in
which IGF-I expression is upregulated and may contribute to the
hyperplasia of smooth muscle that contributes to the formation of
intestinal strictures (53).
Two isoforms of the ribosomal S6K1 are derived from alternative
splicing at the amino terminus: p85S6K1I and p70S6K1
II (35, 36). The p70S6K1
II isoform (p70S6 kinase) is mainly
cytosolic, whereas the p85S6K1
I isoform is predominantly nuclear.
p70S6 kinase, a serine/threonine protein kinase, is activated
by growth factors and plays a central role in cell growth and
proliferation by mediating the phosphorylation of the 40S ribosomal
protein, S6, thereby enabling efficient translation of 5'-terminal
oligopyrimidine tract mRNAs (5-TOPs). This class of mRNAs encodes for
numerous components of the protein synthesis machinery: ribosomal
proteins and elongation factors. A second S6K homologue, S6K2, was
identified in S6K1-deficient mice that exhibited a phenotype of
diminished but not absent growth compared with that of wild-type mice
(42). In S6K1-deficient mice, S6K2 expression was
upregulated and provided partial compensation for the absence of S6K1.
S6K2 also exists as two isoforms, p60S6K2
I and p54S6K2
II. Both
S6K2 isoforms contain a carboxy-terminal nuclear localization signal
not found in S6K1 and are therefore localized predominantly in the nucleus.
Activation of p70S6 kinase depends on the sequential phosphorylation of four sets of serine/threonine sites by several independently regulated kinases (49). The initial step in p70S6 kinase activation involves the phosphorylation of serine/threonine residues in the autoinhibitory pseudosubstrate domain (Ser411, Ser418, Ser424, Ser429, and Thr421) and in the catalytic domain extension (Thr390 and Ser394) under the control of proline-directed kinases (7, 30). The result is the release of the autoinhibitory carboxy-terminal tail from the catalytic domain allowing access to Thr229 and Thr389. Phosphorylation of p70S6 kinase on Thr229 and Thr389 is regulated by the lipid products of PI3K, phosphatidylinositol 3,4,5-trisphosphate (PIP3), and to lesser extent phosphoinositol 4,5-bisphosphate (PIP2), acting both directly on p70S6 kinase and indirectly via 3-phosphoinositide-dependent kinase-1 (PDK-1) (1). Although phosphorylation of p70S6 kinase(Thr389) most closely correlates with p70S6 kinase activity in vivo, considerable positive cooperativity exists with p70S6 kinase(Thr229) phosphorylation (50). Dual phosphorylation of Thr229 and Thr389 fully activates p70S6 kinase.
p70S6 kinase participates in PI3K-dependent regulation of muscle growth by IGF-I in rat and porcine vascular smooth muscle cells (14, 18) and in L6A1 skeletal myoblasts (11). Although several signaling intermediates lying downstream of PI3K have been shown to regulate the activity of p70S6 kinase in other muscle types [PDK-1, Akt, and the mammalian target of rapamycin (mTOR)] it is not known whether IGF-I activates p70S6 kinase in human intestinal smooth muscle cells or the sequence of signaling events emanating from PI3K that regulate p70S6 kinase activity and muscle cell growth.
This study shows that, in human intestinal smooth muscle cells, IGF-I
causes phosphorylation of multiple serine/threonine residues on p70S6
kinase that result in its activation. Serine/threonine residues within
the autoinhibitory domain are phosphorylated in a PI3K-dependent
fashion involving PKC-. p70S6 kinase(Ser389) and
p70S6 kinase(Thr229) in the catalytic domain are
phosphorylated in a PI3K-dependent, PDK-1-dependent fashion. Whereas
IGF-I-induced ERK1/2 and PI3K activation jointly stimulate muscle cell
proliferation, active p70S6 kinase is required for growth to occur
regardless of the pathway involved.
IGF-I also elicits PI3K-dependent, PDK-1-dependent Akt(Ser308) phosphorylation and subsequent Akt(Ser473) autophosphorylation that leads to phosphorylation of mTOR on Ser2448. Although mTOR kinase activity is required for stimulation of cell growth, IGF-I-induced, Akt-dependent mTOR(Ser2448) phosphorylation is regulated independently of mTOR kinase activity and is not required for IGF-I-mediated p70S6 kinase activation and proliferation to occur.
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METHODS |
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Culture of isolated smooth muscle cells from normal human jejunum. Muscle cells were isolated and cultured from the circular muscle layer of human jejunum as described previously (21, 22, 24). Briefly, 4- to 5-cm segments of normal jejunum were obtained from patients undergoing surgery for morbid obesity according to a protocol approved by the University Office of Research Subject Protection. After the segments were opened along the mesenteric border, the mucosa was dissected away and the remaining muscle layer was cut into 2 × 2-cm strips. Slices were obtained separately from the circular layer by using a Stadie-Riggs tissue slicer. The slices were incubated overnight at 37°C in 20 ml of DMEM plus 10% fetal bovine serum (DMEM-10) containing 200 U/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamycin, and 2 µg/ml amphotericin B, to which was added 0.0375% collagenase (CLS type II) and 0.1% soybean trypsin inhibitor. Muscle cells dispersed from the circular layer were harvested by filtration through 500-µm Nitex mesh and centrifuged at 150 g for 5 min. Cells were resuspended and washed twice by centrifugation at 150 g for 5 min. After resuspension in DMEM-10 containing the same antibiotics, the cells were plated at a concentration of 5 × 105 cells/ml as determined by counting in a hemocytometer. Cultures were incubated in a 10% CO2 environment at 37°C. DMEM-10 medium was replaced every 3 days until the cells reached confluence.
Primary cultures of muscle cells were passaged on reaching confluence by first being washed three times with PBS. After the PBS was removed, cells were treated for 2 min with 0.05% trypsin and 0.53 mM EDTA. The trypsin activity was neutralized by addition of a fourfold excess of DMEM-10. The resulting cell suspension was centrifuged at 350 g for 10 min at 4°C. The pellet was resuspended in DMEM-10 at a concentration of 2.5 × 106 cells/ml and plated in the appropriate cultureware. The medium was changed after 24 h. All subsequent studies were performed in first-passage cultured cells after 7 days, at which time the cells were confluent. We have previously shown that these cells express a phenotype characteristic of intestinal smooth muscle as determined by immunostaining for smooth muscle markers and expression of[3H]thymidine incorporation assay. Proliferation of smooth muscle cells in culture was measured by the incorporation of [3H]thymidine as described previously (21, 24, 25). Briefly, the cells were washed free of serum and incubated for 24 h in serum-free DMEM. The quiescent muscle cells were incubated for an additional 24 h with a maximally effective concentration of IGF-I (100 nM) in the presence and absence of various test agents. During the final 4 h of this incubation period, 1 µCi/ml [3H]thymidine was added to the medium. [3H]thymidine incorporation into the perchloric acid extractable pool was used as a measure of DNA synthesis.
Measurement of p70S6 kinase activity by in vitro kinase assay.
The activity of p70S6 kinase was assayed by using an immune complex in
vitro kinase assay by modification of the methods of Zhang et al.
(52). Briefly, confluent muscle cells were rendered quiescent by incubation in serum-free DMEM for 24 h. Cells were stimulated with IGF-I (100 nM) for the indicated periods of time (0-24 h) in the presence or absence of various inhibitors. The reaction was terminated by washing the cells in ice-cold PBS. The cells
were lysed in buffer consisting of (in mM): 50 Tris (pH 7.5), 1 EDTA, 1 EGTA, 0.5 sodium orthovanadate, 50 NaF, 5 sodium pyrophosphate, 10 sodium glycerol phosphate, and 0.1 PMSF, with 0.1% 2-mercaptoethanol,
1% Triton X-100, 1 µg/ml aprotinin, and 1 µg/ml leupeptin. Cell
lysates were clarified by centrifugation, and aliquots of the
supernatant containing equal amounts of protein (200 µg) were
incubated with 4 µg of p70S6 kinase antibody coupled to protein A/G
agarose beads for 2 h at 4°C. The protein A/G agarose-protein immune complexes were washed once with lysis buffer containing 0.5 M
NaCl, twice with lysis buffer alone, and finally in assay buffer
consisting of (in mM): 20 MOPS (pH 7.2), 25 -glycerol phosphate, 5 EGTA, 1 sodium orthovanadate, and 1 dithiothreitol. The resulting
immune complexes were assayed for p70S6 kinase activity in assay
buffer, to which was added (in µM): 15 MgCl2, 100 ATP, 4 PKC inhibitor, 0.4 PKA inhibitor (PKI), 4 Compound R24571, and 50 S6 substrate peptide (AKRRRLSSLRA). The reaction was initiated by the
addition of 10 µCi [
-32P]ATP and continued for 10 min at 30°C. The reaction was terminated by spotting 25-µl aliquots
on P81 phosphocellulose paper. The phosphocellulose paper was washed
three times with 0.75% phosphoric acid and one time with acetone,
dried, transferred to scintillation vials, and counted on a
-scintillation counter. The results were corrected for endogenous
substrates by subtraction of values obtained in the absence of added S6
peptide and for background obtained by immunoprecipitation with
nonimmune serum. Values were expressed in picomoles of phosphate
incorporated into peptide substrate per minute per milligram of protein.
Measurement of PI3K activity by in vitro kinase assay.
PI3K activity was measured as described previously (24).
Briefly, quiescent muscle cells growing in 100-mm dishes were
stimulated for 30 min with IGF-I (100 nM) in the presence and absence
of various inhibitors. The reaction was terminated by rapidly washing cells with 4°C PBS and by lysis of the cells. Lysis buffer consisted of (in mM): 50 Tris · HCl (pH 7.4), 150 NaCl, 1 Na2VO3, 2 EDTA, 1 MgCl2, and 1 CaCl2, with 30 nM leupeptin, 1% trasylol (wt/vol), and 1%
NP-40 (vol/vol). DNA in the lysate was sheared by passage through a
22-gauge needle, and the lysate was incubated for 20 min at 4°C.
Following this incubation, the lysate was centrifuged at 12,000 g for 20 min at 4°C. Aliquots of lysate containing equal amounts of protein were incubated with 25 µl of agarose-conjugated antibody to phosphotyrosine (PY20) with gentle mixing for 2 h at
4°C. The beads were collected by centrifugation at 12,000 g for 5 min at 4°C and washed three times with lysis
buffer and two times with kinase assay buffer. Kinase assay buffer
consisted of (in mM): 50 Tris · HCl (pH 7.8), 50 NaCl, 2 MgCl2, and 0.5 EDTA. After the final washing, the beads
were resuspended in 30 µl of kinase assay buffer to which 10 µl of
sonicated 1 mg/ml phosphatidylinositol was added. The reaction was
initiated by the addition of 5 µl of 50 mM ATP containing 0.5 µCi
[-32P]ATP and was continued for 10 min at 30°C. The
reaction was terminated by addition of 0.5 ml of 1 N HCl and 2 ml of
chloroform-methanol (2:1, vol/vol). Phospholipids were recovered from
the lower organic phase and dried under N2 gas. The dried
phospholipids were dissolved in chloroform and spotted on Silica H gel
TLC plates impregnated with 1% potassium oxalate. Chromatograms were
developed in chloroform-methanol-28% NH3-water
(70:100:15:25, vol/vol). The plates were dried, and the spots
corresponding to authentic PIP3 were visualized and quantitated by using a PhosphorImager (Packard Instruments,
Meridien, CT). Results are expressed as the increase in
[32P] incorporation into PI-3-P above basal values.
Measurement of PDK-1 activity by in vitro kinase assay.
The activity of PDK-1 was measured by an immune complex in vitro kinase
assay according to the methods of Fujita et al. (16). Briefly, quiescent muscle cells were incubated for 30 min with 100 nM
IGF-I. The reaction was terminated by washing cells with ice-cold PBS.
The cells were lysed in buffer consisting of (in mM): 50 Tris (pH 7.5),
1 EDTA, 1 EGTA, 50 NaF, 1 sodium orthovanadate, 5 sodium pyrophosphate,
and 10 sodium -glycerol phosphate, with 0.1% 2-mercaptoethanol
(vol/vol), 0.1% Triton X-100 (vol/vol), and 1 µM Microcystin LR.
Following the addition of 50 µl of protein G agarose, the lysates
were clarified by centrifugation at 4°C. Protein G-antibody complexes
were simultaneously prepared by incubation of 4 µg of anti-PDK-1
antibody (or normal sheep IgG as negative control) with 100 µl of a
50% slurry of protein G agarose beads and 250 µl of lysis buffer
overnight at 4°C. Protein G-antibody complexes were washed twice with
lysis buffer to remove weakly bound antibody by centrifugation at
14,000 rpm. Precleared IGF-I-stimulated cell lysates containing 1 mg of
protein were added to the protein G/antibody complexes and incubated
for 2 h at 4°C to immunoprecipitate active PDK-1. The resulting
immune complexes were washed twice with lysis buffer and twice with
PDK-1 assay buffer. PDK-1 assay buffer consisted of (in mM): 50 Tris · HCl (pH 7.5), 0.1 EDTA, 0.1 EGTA, 10 magnesium acetate,
and 0.1 ATP, with 1% 2-mercaptoethanol (vol/vol), 2.5 µM PKI, and 1 µM Microcystin LR. The resulting PDK-1 immune complexes were
resuspended in 20 µl of PDK-1 assay buffer, and inactive serum- and
glucocorticoid-dependent protein kinase (SGK1;
1-60,
S422D; 500 ng) was added to the immunoprecipitated PDK-1 to be
activated by incubation for 30 min at 30°C. The final assay reaction
was carried out in a total volume of 50 µl. The reaction was
initiated by addition of 4 nmol of Akt/SGK substrate peptide (RPRAATF)
and 10 µCi of [
-32P]ATP and continued for 10 min at
30°C. The reaction was terminated by centrifugation at 14,000 rpm and
spotting of 25-µl aliquots on P81 phosphocellulose paper. The
phosphocellulose paper was washed three times with 0.75% phosphoric
acid and one time with acetone, dried, transferred to scintillation
vials, and counted on a
-scintillation counter. The results were
corrected for endogenous substrates by subtraction of values obtained
after immunoprecipitation with normal sheep IgG. Values were expressed
in picomoles of phosphate incorporated into peptide substrate per
minute per milligram of protein.
Western blot analysis.
The phosphorylation of PDK-1, Akt, mTOR, PKC-, and p70S6 kinase on
specific residues was measured by Western blot analysis by using
standard methods (9, 24, 26). Briefly, confluent muscle
cells were rendered quiescent by incubation for 24 h in serum-free
medium. The cells were stimulated with recombinant human IGF-I for 30 min (the time of peak p70S6 kinase activation). The cells were rapidly
washed with ice-cold PBS and lysed in sample buffer. Lysates were
boiled for 5 min, and equal amounts of total cell protein were
separated with SDS-PAGE under denaturing conditions. After the proteins
were electrotransferred to nitrocellulose, the membranes were incubated
overnight with a 1:1,000-1:2,000 dilution of antibodies
recognizing phosphorylated (activated) signaling intermediates:
PDK-1(Ser241),
PKC-
/
(Thr410/Thr403),
Akt(Ser308), Akt(Ser473),
mTOR(Ser2448), p70S6 kinase(Thr229), p70S6
kinase(Thr389), and p70S6
kinase(Thr421/Ser424). Bands of interest were
visualized with enhanced chemiluminescence. Nitrocellulose membranes
were stripped and reblotted to determine levels of total
(phosphorylated and nonphosphorylated) protein using antibodies
recognizing PDK-1, Akt, mTOR, PKC-
, and p70S6 kinase.
Expression of kinase-inactive Akt. Substitution of methionine for lysine at residue 179 (K179M) results in a kinase-inactive Akt1, which when expressed in cells exerts a dominant-negative effect (43). cDNA for Myc-His-tagged kinase-inactive Akt1(K179M) in the pUSEamp(+) expression vector was purified, and muscle cells growing in six-well plates were transiently transfected with 1 µg of pUSEamp(+)-Akt1(K179M) cDNA or with pUSEamp(+) vector alone as a control by using a LipofectAMINE PLUS reagent kit (Life Technologies). Cells were incubated for 3 h at 37°C with the transfection reagent-DNA complexes. The DNA-containing medium was replaced with DMEM plus 10% FCS. After 48 h incubation, the expression of Akt1(K179M) was confirmed by Western blot analysis using an anti-Myc tag antibody, and the kinase-inactive effect of this mutation on IGF-I signaling was confirmed by analysis of Akt(Ser473) phosphorylation.
Measurement of protein content. The protein content of cell lysates was measured by using the Bio-Rad DC protein assay kit according to manufacturer's directions. Samples were adjusted to provide aliquots of equal protein content before in vitro kinase assay or Western blot analysis.
Statistical analysis. Values given represent the means ± SE of n experiments, where n represents the number of experiments on cells derived from separate primary cultures. Statistical significance was tested by Student's t-test for either paired or unpaired data as appropriate. Analysis of relative densitometric values was performed using ImageJ 1.26t software (National Institutes of Health, Bethesda, MD). Densitometric values for protein bands of phosphorylated signaling intermediates were reported in arbitrary units above background values and normalized to total signaling intermediate protein levels.
Materials.
Recombinant human IGF-I was obtained from Austral Biologicals (San
Ramon, CA); collagenase and soybean trypsin inhibitor were obtained
from Worthington Biochemical (Freehold, NJ); HEPES was obtained from
Research Organics (Cleveland, OH); DMEM and Hank's balanced salt
solution were obtained from Mediatech (Herndon, VA); fetal bovine serum
was obtained from Summit Biotechnologies (Fort Collins, CO);
[-32P]ATP (specific activity 3,000 Ci/mmol),
[3H]thymidine (specific activity 6 Ci/mmol), and
anti-mouse IgG horseradish peroxidase were obtained from NEN (Boston,
MA); rabbit polyclonal antibodies to
phospho-PDK-1(Ser241), phospho-Akt(Ser308),
phospho-Akt(Ser473), phospho-mTOR(Ser2448),
phospho-PKC-
/
(Thr410/Thr403),
phospho-p70S6 kinase(Thr389), phospho-p70S6
kinase(Thr421/Ser424), and anti-rabbit IgG
horseradish peroxidase were obtained from Cell Signaling
Technology (Beverly, MA); rabbit polyclonal antibody to phospho-p70S6
kinase(Thr229) was obtained from R&D Systems (Minneapolis,
MN); agarose-conjugated mouse monoclonal antibody to phosphotyrosine
(PY20) was obtained from Transduction Laboratories (Lexington, KY);
mouse monoclonal antibody to the Myc tag on Akt1(K179M), PDK-1
kinase, and p70S6 kinase assay kits were obtained from Upstate
Biotechnology (Lake Placid, NY); myristoylated PKC-
pseudosubstrate
(myr-PKC-
-PS) was obtained from Biomol (Plymouth Meeting, PA); the
inhibitors LY-294002, U-1026, SB-203580, N
-tosyl-Phe
chloromethyl ketone (TPCK), Microcystin LR, and rapamycin were obtained
from Calbiochem (San Diego, CA); Western blotting materials and DC
protein assay kit were obtained from Bio-Rad Laboratories (Hercules,
CA); and plastic cultureware was obtained from Corning (Corning,
NY). All other chemicals were obtained from Sigma (St Louis, MO).
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RESULTS |
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Role of p70S6 kinase in IGF-I-induced growth. We have previously shown (24) that IGF-I increases [3H]thymidine incorporation into human intestinal muscle cells by joint activation of distinct ERK1/2-dependent and PI3K-dependent pathways. The role of p70S6 kinase in mediating IGF-I-induced growth was examined by measurement of [3H]thymidine incorporation in the presence of the p70S6 kinase inhibitor rapamycin (40). Incubation of quiescent muscle cells with IGF-I (100 nM) for 24 h increased [3H]thymidine incorporation by 392 ± 25% above basal (basal value = 181 ± 9 cpm/µg protein). In the presence of rapamycin (10 nM), IGF-I-induced [3H]thymidine incorporation was abolished (99 ± 2% inhibition). The results imply that IGF-I-induced growth, whether mediated via PI3K-dependent or ERK1/2-dependent mechanisms, requires activation of p70S6 kinase to proceed.
The involvement of potential PI3K-dependent signaling intermediates in IGF-I-induced intestinal smooth muscle cell growth was examined by measuring the effect of selective inhibitors of PDK-1 and PKC-
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IGF-I activates p70S6 kinase. The ability of growth factors to fully stimulate p70S6 kinase activity involves the sequential phosphorylation of distinct sets of serine/threonine residues within p70S6 kinase. Phosphorylation of serine/threonine residues in the autoinhibitory domain of p70S6 kinase (e.g., Thr421 and Ser424) relieves pseudosubstrate inhibition of the catalytic domain and is a prerequisite for phosphorylation of Thr229 in the activation loop of the catalytic domain and of Thr389 in the carboxy-terminal extension of the catalytic domain (1, 30, 49, 50).
Our initial studies showed that p70S6 kinase activity was required for IGF-I to stimulate growth of human intestinal smooth muscle cells. The ability of IGF-I to activate p70S6 kinase in human intestinal muscle cells was therefore examined in two complementary ways. In the first, the ability of IGF-I to elicit phosphorylation of three specific regions of p70S6 kinase (Thr421/Ser424, Thr229, and Thr389) was examined by Western blot analysis using phosphorylation state-specific antibodies. In the second, the ability of IGF-I to stimulate p70S6 kinase activity was measured by using an immune complex in vitro kinase assay. Incubation of human intestinal muscle cells with 100 nM IGF-I for increasing periods of time (0-24 h) elicited time-dependent phosphorylation of p70S6 kinase on Thr421/Ser424, Thr229, and Thr389 that was rapid, occurring within 5 min, was maximal within 30 min (Thr421/Ser424 = 320 ± 25% above basal; Thr229 = 220 ± 20% above basal; Thr389 = 590 ± 40% above basal) and was sustained at submaximal levels for up to 24 h (Fig. 2, A and B). When measured at the 30-min maximum, phosphorylation of Thr421/Ser424, Thr229, and Thr389 elicited by IGF-I was also concentration dependent (Fig. 2C).
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Mechanisms of IGF-I-induced p70S6 kinase activation.
The ability of selective inhibitors of PI3K, PDK-1, and PKC- to
inhibit IGF-I-induced [3H]thymidine incorporation
suggested that these intermediates may participate in the activation of
p70S6 kinase. This was also investigated using the two complementary
techniques: p70S6 kinase phosphorylation on residues
Thr421/Ser424, Thr229, and
Thr389 was measured by Western blot analysis, and
activation of p70S6 kinase was measured by using in vitro kinase assay.
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PI3K-dependent signaling intermediates activated by IGF-I.
The pattern of inhibition of IGF-I-induced p70S6 kinase
phosphorylation, p70S6 kinase activation, and growth suggested that PI3K and PDK-1 play essential roles in these processes, as does PKC-, albeit to a lesser extent. The sequence of events initiated by
IGF-I was therefore also investigated by using several complementary techniques. Activation of PI3K and PDK-1 in response to IGF-I was
measured by using the in vitro kinase assay as had been done for p70S6
kinase. IGF-I stimulated phosphorylation of signaling intermediates:
PDK-1(Ser248), Akt(Ser473), and
mTOR(Ser2448) were measured by using phosphospecific
antibodies in Western blot analysis. The sequence of signaling events
initiated by IGF-I was then determined with the use of selective inhibitors.
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Effects of expression of a kinase-inactive Akt. Our initial studies showed that, although IGF-I elicited PI3K-dependent and PDK-1-dependent phosphorylation of Akt(Ser473) (Fig. 5 and Table 2), a kinase-inactive Akt mutant did not affect IGF-I induced, PI3K-dependent proliferation (Fig. 1B). The mechanisms leading to IGF-I-induced activation of Akt and the participation of Akt in IGF-I-induced p70S6 kinase activation were examined further in human intestinal muscle cells transfected with kinase-inactive Akt1(K179M) or empty vector.
The expression of Akt1(K179M) was confirmed by identification of the Myc tag by Western blot analysis (Fig. 6A). The ability of kinase-inactive Akt1(K179M) to block IGF-I-induced Akt(Ser473) phosphorylation was confirmed by incubation of quiescent muscle cells for 30 min with 100 nM IGF-I and measurement of Akt(Ser473) phosphorylation by Western blot analysis. IGF-I-elicited phosphorylation of Akt(Ser473) (vector = 265 ± 31% above basal) was inhibited 87 ± 3% in cells transfected with kinase-inactive Akt1(K179M) (Fig. 6, A and B). Interestingly, in contrast to phosphorylation of Akt on Ser473, IGF-I-induced phosphorylation of Akt on Ser308 was not affected by expression of the kinase-inactive Akt1(K179M) mutant [vector = 315 ± 40% above basal; Akt1(K179M) = 360 ± 25% above basal]. The results provide evidence that the phosphorylation of Akt(Ser473) required the intrinsic kinase activity of Akt(Leu179), whereas phosphorylation of Akt(Ser308) occurred upstream of the intrinsic Akt kinase activity resident within Akt(Leu179).
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DISCUSSION |
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IGF-I initiates a sequence of signaling events that culminates in
cellular proliferation. A key component of this process is the
activation of p70S6 kinase. p70S6 kinase regulates phosphorylation of
the 40S ribosomal protein S6, a process that enables efficient translation of 5-TOPs and progression through the G1 phase
of the cell cycle. We have previously shown that IGF-I stimulates proliferation of human intestinal smooth muscle cells (22,
24) by activating both PI3K and ERK1/2. In these cells, these
two pathways are distinct and jointly mediate the effects of IGF-I on
growth. Phosphorylation of PI3K by IGF-I results in the production of
PIP3 (24). PIP3, the major lipid
product of activated PI3K (and to a lesser extent PIP2),
regulates both the localization, by stimulating plasma membrane
association, and the activation, by facilitating phosphorylation, of
downstream signaling molecules such as PDK-1, Akt, and PKC- that
leads to p70S6 kinase phosphorylation and activation.
In the present study, the PI3K-dependent signaling events activated by
IGF-I that regulate p70S6 kinase activity and cell proliferation have
been elucidated. IGF-I causes the sequential phosphorylation of PI3K,
PDK-1, Akt, and mTOR. PI3K and PDK-1 activation mediate IGF-I-induced
p70S6 kinase activation and regulate proliferation. Akt activation via
PDK-1, although sufficient to modestly increase the levels of
mTOR(Ser2448) phosphorylation, is not required for
IGF-I-induced p70S6 kinase activation or growth to occur. Moreover,
mTOR kinase activity is required for IGF-I-induced cell proliferation
but is distinct from the state of mTOR(Ser2448)
phosphorylation and is not regulated by Akt. IGF-I-induced, PI3K-dependent activation of the atypical PKC isoform PKC- also contributes to p70S6 kinase activation and growth.
The evidence supporting the sequential activation of PI3K, PDK-1, and
PKC- leading to IGF-I-induced p70S6 kinase activation and
proliferation can be summarized as follows: 1) activation of
PI3K was blocked by the PI3K inhibitor but not affected by the PDK-1,
PKC-
, or p70S6 kinase antagonists; 2) both the
phosphorylation and activation of PDK-1 were abolished by the PI3K
inhibitor or the PDK-1 inhibitor and were inhibited by the PKC-
inhibitor but were not affected by the p70S6 kinase antagonist or
expression of kinase-inactive Akt; 3) phosphorylation of
PKC-
was blocked by the PI3K inhibitor and the PDK-1 inhibitor but
was not affected by the p70S6 kinase antagonist or kinase-inactive Akt;
4) phosphorylation of p70S6
kinase(Thr421/Ser424) was blocked by the PI3K
inhibitor and the p70S6 kinase inhibitor and was partially inhibited by
the PKC-
inhibitor but was not affected by the PDK-1 inhibitor or
kinase-inactive Akt; 5) phosphorylation of p70S6
kinase(Thr229) and p70S6
kinase(Thr389), activation of p70S6 kinase, and
cellular proliferation were blocked by the PI3K, PDK-1, and p70S6
kinase inhibitors and were partially inhibited by the PKC-
inhibitor
but were not affected by kinase-inactive Akt.
PDK-1, a serine/threonine kinase, links the activation of PI3K and Akt. PDK-1 can also phosphorylate and activate other PI3K-dependent effectors: atypical PKC isoforms and p70S6 kinase. PDK-1 was initially thought to be constitutively active, associated with the plasma membrane, and not subject to growth factor (including IGF-I)-induced phosphorylation or activation (12). The findings of the current study corroborate and extend more recent information showing that insulin (10) and IGF-I (this study) can induce the phosphorylation of PDK-1 on Ser241. Mutants of PDK-1 with disrupted pleckstrin homology domains that do not localize to the plasma membrane, however, are not activated by growth factors (10).
Binding of growth factor-stimulated PIP3 to the pleckstrin homology domain of Akt causes translocation of Akt to the plasma membrane, relieves steric inhibition, and exposes the activation loop of Akt to PDK-1-mediated phosphorylation of Akt(Ser308) (2). Activation of Akt, however, requires the phosphorylation of both Ser308 and Ser473 in the carboxy terminus. The exact mechanism of Akt(Ser473) phosphorylation, termed the PDK-2 site, and the identity of PDK-2 have remained elusive. Recently, integrin-linked kinase (ILK) has been identified as a putative PDK-2 (3). Whether ILK regulates Akt(Ser473) phosphorylation in human intestinal smooth muscle cells is unknown. Alternative mechanisms for Akt(Ser473) phosphorylation have been proposed. Evidence suggesting that Akt(Ser473) is autophosphorylated by an intrinsic Akt kinase activity that is stimulated by PDK-1-dependent Akt(Ser308) phosphorylation was derived from studies in which kinase-inactive Akt1(K179M) was expressed in human embryonic kidney cells, (HEK-293E cells), Hep G2 cells (47), and Chinese hamster ovary cells (51). This mechanism of Akt activation is supported by the findings of the current paper: when the effects of PDK-1 are blocked by TPCK, the ability of IGF-I to elicit phosphorylation of Akt(Ser308) or Akt(Ser473) is abolished. In contrast, expression of kinase-inactive Akt1(K179M) in human intestinal smooth muscle does not affect IGF-I-induced Akt(Ser308) phosphorylation but does abolish Akt(Ser473) phosphorylation, implying that the autophosphorylation of Akt following IGF-I-induced Akt(Ser308) phosphorylation is required for Akt(Ser473) phosphorylation to occur. Other kinases have been shown to regulate Akt activity, e.g., p38 MAPK-dependent Akt(Ser473) phosphorylation occurs in human neutrophils (34). These mechanisms do not appear to operate in human intestinal smooth muscle cells, because the p38 MAPK and the MKK1/2 inhibitors do not affect the ability of IGF-I to phosphorylate Akt. Interestingly, activation of Akt does not appear to be a requirement for IGF-I-mediated p70S6 kinase activation or proliferation of human intestinal muscle cells. In cells expressing kinase-inactive Akt1(K179M), IGF-I retained its ability to activate p70S6 kinase and to stimulate proliferation. Activation of Akt by IGF-I in human intestinal muscle cells likely regulates other cellular processes, such as inhibition of apoptosis, a process that has also been observed in this and other cell types (23, 27).
The homologous atypical PKC isoforms PKC- and PKC-
are downstream
targets in the PI3K pathway. PI3K-dependent activation of PKC-
occurs through three interrelated mechanisms: PDK-1-dependent phosphorylation of PKC-
on Thr410 in the activation
loop, autophosphorylation of Thr560, and
phosphorylation-independent conformational relief of pseudosubstrate inhibition (perhaps via interaction with PIP3)
(44). Evidence that PKC-
represents a physiological
target of PDK-1 comes from PDK-1-deficient embryonic stem cells that
show markedly decreased levels of conventional (PKC-
, PKC-
1,
PKC-
) and novel (PKC-
, PKC-
) PKC isoforms compared with
PDK-1-positive cells (4). In contrast, levels of atypical
PKC-
are unchanged, but PKC-
cannot be phosphorylated on
Thr410. IGF-I-mediated proliferation has been shown to
involve the atypical PKC isoform PKC-
in rat adipocytes
(45) and in the rat clonal
-cell line RIN 1046-38
(19). In the present study, IGF-I elicited phosphorylation
of atypical PKC-
on Thr410 of the activation loop and/or
PKC-
on the homologous residue Thr403. Activation of
PKC-
/
by IGF-I participated in p70S6 kinase activation and
proliferation of muscle, and these effects were inhibited by the
cell-permeant PKC-
pseudosubstrate inhibitor myr-PKC-
-PS
(which also blocks the effects of PKC-
). These two atypical PKC
isoforms appear to act interchangeably in their ability to mediate
PI3K-dependent cellular processes such as Glut4 translocation in
response to insulin or serum-induced activation of p70S6 kinase (6). This is not to say that conventional and novel PKC
isoforms are not regulated by PDK-1 (see above) or do not
contribute to the complex mechanism of p70S6 kinase activation and
regulation of proliferation.
mTOR is a constitutively active kinase that plays a permissive role in
regulating p70S6 kinase activity and thus the ribosomal biogenesis
necessary for cell division. mTOR senses the level of amino acids and
ATP that are available to the cell (13, 38). The current
study shows that basal levels of mTOR(Ser2448)
phosphorylation can be augmented by IGF-I via an Akt-dependent mechanism as has been shown for insulin (37).
Akt-dependent mTOR(Ser2448) phosphorylation does not appear
to regulate p70S6 kinase activity, however. Other investigators have
shown that mutation of mTOR(Ser2448) to the
nonphosphorylated alanine, stimulation with mitogens, withdrawal of
amino acids, or treatment with rapamycin all have little affect on mTOR
kinase activity (17, 38). Deletion of the domain
surrounding Ser2448 in mTOR (amino acids
2430-2450), in fact, increases p70S6 kinase activity, suggesting
that a repressor function for mTOR resides within this region
(41). The rapamycin-FKBP12 gain-of-function complex
inhibits signaling downstream of mTOR by disinhibiting the effects of
mTOR on protein phosphatase 2A (PP2A) (33), which is
normally associated with p70S6 kinase and in an inactive form. Once
activated, PP2A rapidly dephosphorylates numerous residues of p70S6
kinase with Thr389 dephosphorylation, most closely
paralleling the loss of p70S6 kinase activity (32).
Although addition of rapamycin to human intestinal smooth muscle cells
did affect IGF-I-induced mTOR activity, this effect was a minor one.
Similar effects of rapamycin have been observed in 3T3 L1 adipocytes
treated with insulin (39). mTOR has also been shown to
directly phosphorylate p70S6 kinase on Thr389 in
HEK-293 cells (8). The present study supports the
view that mTOR functions in a coregulatory fashion with the
PI3K-dependent signaling pathway involving PDK-1 and PKC-
activation. Activation of both pathways is required for p70S6 kinase
activation and cell proliferation to occur.
In summary, the present study shows that, in human intestinal smooth
muscle cells, IGF-I activates a sequence of events consisting of
initial PI3K activation followed by PDK-1 and PKC- phosphorylation and activation that leads to phosphorylation of specific residues within p70S6 kinase that result in full p70S6 kinase activation and
proliferation. PI3K, perhaps via PKC-
/
, regulates phosphorylation of serine/threonine residues in the autoinhibitory domain of p70S6 kinase. PI3K-dependent activation of PDK-1 and PDK-1-dependent PKC-
/
activation, in turn, regulate phosphorylation of
serine/threonine residues in the catalytic domain of p70S6 kinase,
activation of p70S6 kinase, and muscle cell growth. IGF-I-induced Akt
activation, although sufficient to increase mTOR(Ser2448)
phosphorylation, does not significantly contribute to the regulation of
mTOR kinase activity and is not required for either IGF-I-induced p70S6
kinase activation or cell proliferation to occur.
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Institutes of Diabetes, Digestive, and Kidney Diseases Grant DK-49691.
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: J. F. Kuemmerle, Division of Gastroenterology, Virginia Commonwealth Univ., PO Box 980711, Richmond, VA 23298-0711 (E-mail: jkuemmerle{at}hsc.vcu.edu).
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.
First published November 20, 2002;10.1152/ajpgi.00310.2002
Received 29 July 2002; accepted in final form 19 November 2002.
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