Phosphorylation Of Phosphoinositide-dependent Kinase-1 Research Articles

Chrysin enhances anticancer drug-induced toxicity mediated by the reduction of claudin-1 and 11 expression in a spheroid culture model of lung squamous cell carcinoma cells

The aberrant expression of claudins (CLDNs), which are tight junctional proteins, is seen in various solid tumors, but the regulatory mechanisms and their pathophysiological role are not well understood. Both CLDN1 and CLDN11 were highly expressed in human lung squamous cell carcinoma (SCC). Chrysin, found in high concentration in honey and propolis, decreased CLDN1 and CLDN11 expression in RERF-LC-AI cells derived from human lung SCC. The phosphorylation level of Akt was decreased by chrysin, but those of ERK1/2 and c-Jun were not. LY-294002, an inhibitor of phosphatidylinositol 3-kinase, inhibited the phosphorylation of Akt and decreased the expression levels of CLDN1 and CLDN11. The association between phosphoinositide-dependent kinase 1 (PDK1) and Akt was inhibited by chrysin, but the phosphorylation of PDK1 was not. Immunoprecipitation and quartz-crystal microbalance assays revealed that biotinylated-chrysin binds directly to Akt. The knockdown of CLDN1 and CLDN11 using small interfering RNAs increased the transepithelial flux of doxorubicin (DXR), an anthracycline anticancer drug. Similarly, both chrysin and LY-294002 increased DXR flux. Neither CLDN1 knockdown, CLDN11 knockdown, nor chrysin changed the anticancer drug-induced cytotoxicity in a two-dimensional culture model, whereas they enhanced cytotoxicity in a spheroid culture model. Taken together, chrysin may bind to Akt and inhibit its phosphorylation, resulting in the elevation of anticancer drug-induced toxicity mediated by reductions in CLDN1 and CLDN11 expression in RERF-LC-AI cells. We suggest that chrysin may be useful as an adjuvant chemotherapy in lung SCC.

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction.

Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with μ-conotoxin GIIIB (μ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release.

Periostin suppresses in vivo invasiveness via PDK1/Akt/mTOR signaling pathway in a mouse orthotopic model of bladder cancer.

Periostin is an extracellular matrix protein involved in the regulation of intercellular adhesion. The present study investigated the in vivo tumor suppressor function of periostin in a mouse orthotopic model of bladder cancer. Retroviral vectors were used to transfect human bladder cancer UMUC-3 cell line with periostin. Bladders of nude mice that were transurethrally instilled with periostin-expressing UMUC-3 cells were revealed to weigh less compared with bladders instilled with vector control cells. In total, five (83.3%) of six vector control UMUC-3 bladder tumors exhibited histological evidence of muscle invasion. However, none of the five periostin-expressing UMUC-3 bladder tumors revealed muscle invasion. Thick edematous lesions were present in the submucosa of periostin-expressing UMUC-3 bladder tumors. The expression of periostin also suppressed in vitro cell invasiveness of UMUC-3 cells without affecting cellular proliferation. The level of phosphorylation of phosphoinositide-dependent kinase-1 (PDK1), protein kinase B (Akt) and S6 ribosomal protein, a downstream protein of mammalian target of rapamycin (mTOR) was decreased in periostin-expressing UMUC-3 cells compared with vector control cells. Treatment with 100 ng/ml recombinant human periostin protein also suppressed cell invasiveness and phosphorylation of PDK1, Akt and S6 in UMUC-3 cells, consistent with results using periostin-expressing UMUC-3 cells. Treatment with PDK1, Akt and mTOR inhibitors significantly suppressed UMUC-3 cell invasiveness. These results demonstrate that periostin suppresses in vivo and in vitro invasiveness of bladder cancer via the PDK1/Akt/mTOR signaling pathway. Periostin may be useful as a potent chemotherapeutic agent by suppressing bladder cancer invasiveness.

P5-06-04: Mechanisms of Acquired Resistance to Insulin-Like Growth Factor 1 Receptor Inhibitor in MCF-7 Breast Cancer Cell Line.

Abstract Background: No studies have yet clarified the mechanism of acquired resistance to insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitor (TKI). Our previous study of 16 breast cancer cell lines found that only MCF-7 expressed high levels of insulin receptor substrate (IRS)-1 and was sensitive to the IGF-1R-TKI NVP-AEW541. Material and Methods: We developed a model of acquired resistance to NVP-AEW541 by continuously exposing MCF-7 cells to NVP-AEW541, naming the model MCF-7-NR. To explore the mechanism of acquired resistance to NVP-AEW541, the effects of NVP-AEW541 on cell growth and IGF-1R signaling in MCF-7 and MCF-7-NR cells were examined. Results: With Western blot analysis, we found that MCF-7-NR had much lower levels of IRS-1 than parental MCF-7. While phosphorylation of Akt was completely inhibited by administration of NVP-AEW541 (3 μM) in both cell lines, phosphorylation of S6K remained high only in MCF-7-NR. The notion of Akt-independent S6K phosphorylation in MCF-7-NR was further supported by the fact that cell growth and phosphorylation of S6K was affected by administration of the Akt inhibitor perifosine to a lesser degree in MCF-7-NR than in MCF-7. Further, the mTOR inhibitor everolimus inhibited phosphorylation of S6K and cell growth equally in both lines. Screening of MCF-7 and MCF-7-NR lines for phosphorylation of 42 receptor tyrosine kinases with and without 3μM NVP-AEW541 showed that Tyro3 phosphorylation remained high only in MCF-7-NR cells. Gene silencing of Tyro3 using siRNA resulted in reduced cell growth, decreased phosphorylation of phosphoinositide-dependent kinase-1 (PDK-1) and protein kinase C α/βII, reduced expression of cyclin D1 in the MCF-7-NR line, with minimal effects evident in the MCF-7 line. Discussion: Findings from our current study support the possibility of predicting sensitivity to NVP-AEW541 by measuring IRS-1 expression, as hypothesized in our previous study. Akt-independent activation of mTOR/S6K by an as-yet-undefined mechanism appears to induce acquired resistance to NVP-AEW541, and as such mTOR inhibitors may have therapeutic value. Tyro3 upregulation and migration of control of cellular growth and cyclin D1 expression may also induce resistance to NVP-AEW541. Although the validity of these findings should be evaluated in further preclinical studies and eventually in clinical trials, our observations may lead to individualized use of NVP-AEW541 and the development of “backup drugs” against tumors that acquire resistance to NVP-AEW541. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-04.

S6K inhibition renders cardiac protection against myocardial infarction through PDK1 phosphorylation of Akt

In the present study, we observed a rapid and robust activation of the ribosomal protein S6K (S6 kinase) provoked by MI (myocardial infarction) in mice. As activation of S6K promotes cell growth, we hypothesized that increased S6K activity contributes to pathological cardiac remodelling after MI and that suppression of S6K activation may prevent aberrant cardiac remodelling and improve cardiac function. In mice, administration of rapamycin effectively suppressed S6K activation in the heart and significantly improved cardiac function after MI. The heart weight/body weight ratio and fibrotic area were substantially reduced in rapamycin-treated mice. In rapamycin-treated mice, decreased cardiomyocyte remodelling and cell apoptosis were observed compared with vehicle-treated controls. Consistently, inhibition of S6K with PF-4708671 displayed similar protection against MI as rapamycin. Mechanistically, we observed significantly enhanced Thr308 phosphorylation and activation of Akt in rapamycin- and PF-4708671-treated hearts. Cardiomyocyte-specific deletion of PDK1 (phosphoinositide-dependent kinase 1) and Akt1/3 abolished cardioprotection after MI in the presence of rapamycin administration. These results demonstrate that S6K inhibition rendered beneficial effects on left ventricular function and alleviated adverse remodelling following MI in mice by enhancing Akt signalling, suggesting the therapeutic value of both rapamycin and PF-4708671 in treating patients following an MI.

Abstract 3578: A novel and selective 3-phosphoinositide-dependent kinase-1 inhibitor, PF-5177624, blocks insulin-like growth factor-1 induced tumorigenesis in breast cancer cells

Abstract Phosphatidylinositol 3-kinases (PI3Ks) phosphorylate membrane phosphatidylinositides to generate phosphatidylinositol 3,4,5-triphosphate (PIP3); PIP3 serves as an important secondary messenger recruiting and activating proteins that contain a pleckstrin homology (PH) domain including AKT and 3’-phosphoinositide-dependent kinase-1 (PDK1). PDK1 is a 63-kDa Ser/Thr kinase with a catalytic domain near its N-terminus and a pleckstrin homology domain at its C-terminus. The PH domain is necessary for targeting PDK1 to the plasma membrane to phosphroylate the T-loop sites of numerous substrates such as AKT at the residue threonine-308 (T308). The phosphorylation at both T308 and the serine-473 residue by mTORC2, fully activates AKT to induce the downstream signaling pathways that are important for tumor progression. PDK1 also has been shown to phosphorylate p70S6K, isoforms of PKCs, and many other kinase substrates. The oncogenic activity of aberrant PI3K pathway signaling through PDK1 has been extensively validated. PDK1 is highly expressed in a majority of human breast cancers and cell lines, and elevated phosphorylation of PDK1 was associated with PIK3CA mutations in human breast tumors and cell lines. Therefore, targeting PDK1 in the PI3K/AKT pathway may provide an opportunity for breast cancer treatment. In this study, we demonstrate that insulin-like growth factor-1 (IGF-1) stimulates PDK1 activity and a specific and potent PDK1 inhibitor, PF-5177624, inhibits IGF-1 stimulated downstream signaling in breast cancer cells, including a reduction in phosphorylation of both AKT (T308) and p70S6K. The inhibition of PDK1 activity through the IGF-1 axis is sufficient to induce in vitro anti-tumor activity in breast cancer cells such that PF-5177624 inhibits cell proliferation and cell transformation in breast cancer cells. Our data suggest that a specific and potent PDK1 inhibitor (PF-5177624) is likely to have antitumor activity in breast cancer cells and can be developed as a cancer drug for breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3578. doi:10.1158/1538-7445.AM2011-3578

Selective Disruption of Insulin-like Growth Factor-1 (IGF-1) Signaling via Phosphoinositide-dependent Kinase-1 Prevents the Protective Effect of IGF-1 on Human Cancer Cell Death

Insulin-like growth factor-1 (IGF-1) signaling system exerts a broad antiapoptotic function and plays a crucial role in resistance to anticancer therapies. Exposure of MCF-7 breast cancer cells to IGF-1 rapidly and transiently induced tyrosine phosphorylation and activation of phosphoinositide-dependent kinase-1 (PDK1). This was paralleled by Akt/protein kinase B and protein kinase C-zeta phosphorylation, at Thr(308) and Thr(410), respectively. IGF-1 treatment also enhanced PDK1 interaction with IGF-1 receptor (IGF-1R) in intact MCF-7 cells. Pulldown assays revealed that PDK1 bound IGF-1R in vitro and that the region encompassing amino acids 51-359 of PDK1 was necessary for the interaction. Synthetic peptides corresponding to IGF-1R C terminus amino acids 1295-1337 (C43) and to PDK1 amino acids 114-141 reduced in vitro IGF-1R/PDK1 interaction in a concentration-dependent manner. Loading of fluoresceinated-C43 (fluorescein isothiocyanate (FITC)-C43) into MCF-7 cells significantly reduced IGF-1R/PDK1 interaction and phosphorylation of PDK1 substrates. Moreover, FITC-C43 intracellular loading reverted the protective effect of IGF-1 on growth factor deprivation-induced cell death. Finally, the inhibition of IGF-1R/PDK1 interaction and signaling by FITC-C43 was accompanied by 2-fold enhanced killing capacity of cetuximab in human GEO colon adenocarcinoma cells and was sufficient to restore cell death in cetuximab-resistant cell clones. Thus, disruption of PDK1 interaction with IGF-1R reduces IGF-1 survival effects in cancer cells and may enhance cell death by anticancer agents.

Aging-Associated Dysfunction of Akt/Protein Kinase B: S-Nitrosylation and Acetaminophen Intervention

BackgroundAged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s) underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB) is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention.Principal FindingsCompared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308) was higher in soleus muscles of very aged rats (33-months). Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR) phosphorylation, along with decreased levels of insulin receptor beta (IR-β), phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1) (Ser241). In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS). Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month) animals with acetaminophen (30 mg/kg body weight/day) for 6-months.ConclusionsThese data demonstrate that Akt dysfunction and increased S-nitrosylation of Akt may contribute to age-associated disorders in skeletal muscle and that acetaminophen may be efficacious for the treatment of age-related muscle dysfunction.

The kinase PDK1 integrates T cell antigen receptor and CD28 coreceptor signaling to induce NF-κB and activate T cells

In addition to T cell receptor (TCR) ligation, activation of CD28 coreceptor by costimulatory molecule B7 is required for transcription factor NF-κB induction and robust T cell activation, though exactly how CD28 contributes to this remains incompletely understood. We demonstrated here that phosphoinositide-dependent kinase 1 (PDK1) plays an essential role in integrating TCR and CD28 signals. Upon deletion of PDK1 in T cells, TCR-CD28 signals failed to induce NF-κB activation or protein kinase C θ (PKC-θ) phosphorylation, although T cell survival and pathways dependent on p38 and Jnk kinases or transcription factor NF-AT were unaffected. CD28 facilitated NF-κB activation by regulating PDK1 recruitment and phosphorylation, which are necessary for efficient binding of PDK1 to PKC-θ and CARMA1, and thus for NF-κB induction.

The basal level of intracellular calcium gates the activation of phosphoinositide 3‐kinase‐Akt signaling by brain‐derived neurotrophic factor in cortical neurons

Brain-derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase-Akt pathway. Although previous studies suggested the roles of mitogen-activated protein kinase, phospholipase C-gamma-mediated intracellular calcium ([Ca2+]i) increase, and extracellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only phosphoinositide 3-kinase, but not phospholipase C and mitogen-activated protein kinase activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extracellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N '-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA-AM and W13-treated neurons. We further demonstrated that the phosphorylation of phosphoinositide-dependent kinase-1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling.

Targeting the signaling pathway of acylation stimulating protein

Acylation stimulating protein (ASP; C3adesArg) stimulates triglyceride synthesis (TGS) and glucose transport in preadipocytes/adipocytes through C5L2, a G-protein-coupled receptor. Here, ASP signaling is compared with insulin in 3T3-L1 cells. ASP stimulation is not Galpha(s) or Galpha(i) mediated (pertussis and cholera toxin insensitive), suggesting G(alphaq) as a candidate. Phospholipase C (PLC) is required, because the Ca(2+) chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester and the PLC inhibitor U73122 decreased ASP stimulation of TGS by 93.1% (P < 0.0.001) and 86.1% (P < 0.004), respectively. Wortmannin and LY294002 blocked ASP effect by 69% (P < 0.001) and 116.1% (P < 0.003), respectively, supporting phosphatidylinositol 3-kinase (PI3K) involvement. ASP induced rapid, transient Akt phosphorylation (maximal, 5 min; basal, 45 min), which was blocked by Akt inhibition, resembling treatment by insulin. Downstream of PI3K, mamalian target of rapaycin (mTOR) is required for insulin but not ASP action. By contrast, both ASP and insulin activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK(1/2)) pathway, with rapid, pronounced increases in ERK(1/2) phosphorylation, effects partially blocked by PD98059 (64.7% and 65.9% inhibition, respectively; P < 0.001). Time-dependent (maximal, 30 min) transient calcium-dependent phospholipase A(2) (cPLA(2))(-Ser505) phosphorylation (by MAPK/ERK(1/2)) was demonstrated by Western blot analysis. ASP signaling involves sequential activation of PI3K and PLC, with downstream activation of protein kinase C, Akt, MAPK/ERK(1/2), and cPLA(2), all of which leads to an effective and prolonged stimulation of TGS.

Developmental Regulation of Protein Kinase B Activation Is Isoform Specific in Skeletal Muscle of Neonatal Pigs

The postprandial activation of the insulin signaling pathway that leads to translation initiation is enhanced in skeletal muscle of the neonate and decreases with development in parallel with the developmental decline in muscle protein synthesis. Our previous study showed that the activity of protein kinase B (PKB), a major insulin signaling component, was higher in 7- than in 26-d-old pigs. To examine the molecular mechanisms involved, we determined PKB isoform abundance and phosphorylation state, the abundance of its kinases, and PKB's association with its kinases. The abundances of total PKB, PKBalpha, and PKBgamma were higher in muscle of 7- than in 26-d-old pigs whereas PKBbeta abundance was similar in the two age groups. PKB phosphorylation at Thr308 was higher in 7- than in 26-d-old pigs but PKB phosphorylation at Ser473 was similar in both age groups. The association of PKB with 3'-phosphoinositide-dependent kinase-1 (PDK-1), a kinase that phosphorylates PKB at Thr308, and PDK-1 abundance were higher in 7- than in 26-d-old pigs. Moreover, PDK-1 phosphorylation at Ser-241, a site that is crucial for PDK-1 activation, was higher in 7- than in 26-d-old pigs. However, the association of PKB with integrin-linked kinase (ILK), a kinase that potentially phosphorylates PKB at Ser473, and ILK abundance were similar in both age groups. The result suggests that the developmental change in PKB activation is isoform specific and involves regulation by PDK-1.

RET/PTC (rearranged in transformation/papillary thyroid carcinomas) tyrosine kinase phosphorylates and activates phosphoinositide-dependent kinase 1 (PDK1): an alternative phosphatidylinositol 3-kinase-independent pathway to activate PDK1.

Thyroid cancers are a leading cause of death due to endocrine malignancies. RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although the oncogenic potential of RET/PTC is related to intrinsic tyrosine kinase activity, the substrates for this enzyme are yet to be identified. In this report, we show that phosphoinositide-dependent kinase 1 (PDK1), a pivotal serine/threonine kinase in growth factor-signaling pathways, is a target of RET/PTC. RET/PTC and PDK1 colocalize in the cytoplasm. RET/PTC phosphorylates a specific tyrosine (Y9) residue located in the N-terminal region of PDK1. Y9 phosphorylation of PDK1 by RET/PTC requires an intact catalytic kinase domain. The short (iso 9) and long forms (iso 51) of the RET/PTC kinases (RET/PTC1 and RET/PTC3) induce Y9 phosphorylation of PDK1. Moreover, Y9 phosphorylation of PDK1 by RET/PTC does not require phosphatidylinositol 3-kinase or Src activity. RET/PTC-induced phosphorylation of the Y9 residue results in increased PDK1 activity, decrease of cellular p53 levels, and repression of p53-dependent transactivation. In conclusion, RET/PTC-induced tyrosine phosphorylation of PDK1 may be one of the mechanisms by which it acts as an oncogenic tyrosine kinase in thyroid carcinogenesis.

Phosphorylation of the Catalytic Subunit of Protein Kinase A: AUTOPHOSPHORYLATION VERSUSPHOSPHORYLATION BY PHOSPHOINOSITIDE-DEPENDENT KINASE-1

The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase. It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (), but the Escherichia coli-expressed C-subunit undergoes autophosphorylation. To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit. Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1. Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194). Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus. To address the in vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies. The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible for in vivo phosphorylation of PKA.

Regulation of novel protein kinase C epsilon by phosphorylation.

The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKCepsilon isoform, these are Thr(566) in the activation loop, Thr(710) in the turn motif and Ser(729) in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKCepsilon kinase activity in controlling the phosphorylation of Thr(566) and Ser(729). In NIH 3T3 fibroblasts, PKCepsilon migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr(566) and Ser(729). Cells transfected with an active PDK-1 allele also resulted in increased PKCepsilon Thr(566) and Ser(729) phosphorylation, whereas an active myristoylated PKCepsilon mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKCepsilon were not phosphorylated at Ser(729) in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr(566), an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKCepsilon in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P(3), concomitant with an increase in Ser(729) phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.

Regulation of novel protein kinase C ε by phosphorylation

The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKC∊ isoform, these are Thr566 in the activation loop, Thr710 in the turn motif and Ser729 in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKC∊ kinase activity in controlling the phosphorylation of Thr566 and Ser729. In NIH 3T3 fibroblasts, PKC∊ migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr566 and Ser729. Cells transfected with an active PDK-1 allele also resulted in increased PKC∊ Thr566 and Ser729 phosphorylation, whereas an active myristoylated PKC∊ mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKC∊ were not phosphorylated at Ser729 in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr566, an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKC∊ in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P3, concomitant with an increase in Ser729 phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.

Insulin stimulates increased catalytic activity of phosphoinositide-dependent kinase-1 by a phosphorylation-dependent mechanism.

Phosphoinositide-dependent kinase-1 (PDK-1) is a serine-threonine kinase downstream from PI 3-kinase that phosphorylates and activates other important kinases such as Akt that are essential for cell survival and metabolism. Previous reports have suggested that PDK-1 has constitutive catalytic activity that is not regulated by stimulation of cells with growth factors. We now show that insulin stimulation of NIH-3T3(IR) cells or rat adipose cells may significantly increase the intrinsic catalytic activity of PDK-1. Insulin treatment of NIH-3T3(IR) fibroblasts overexpressing PDK-1 increased both phosphorylation of recombinant PDK-1 in intact cells and PDK-1 kinase activity in an immune-complex kinase assay. Insulin stimulation of rat adipose cells also increased catalytic activity of endogenous PDK-1 immunoprecipitated from the cells. Both insulin-stimulated phosphorylation and activity of PDK-1 were inhibited by wortmannin and reversed by treatment with the phosphatase PP-2A. A mutant PDK-1 with a disrupted PH domain (W538L) did not undergo phosphorylation or demonstrate increased kinase activity in response to insulin stimulation. Similarly, a PDK-1 phosphorylation site point mutant (S244A) had no increase in kinase activity in response to insulin stimulation. Thus, the insulin-stimulated increase in PDK-1 catalytic activity may involve PI 3-kinase- and phosphorylation-dependent mechanisms. We conclude that the basal constitutive catalytic activity of PDK-1 in NIH-3T3(IR) cells and rat adipose cells can be significantly increased upon insulin stimulation.