ANTICANCER RESEARCH 37 : 5415-5423 (2017) doi:10.21873/anticanres.11969

Modulation of PI3K/PTEN Pathway Does Not Affect Catalytic Activity of PDK1 in Jurkat Cells KEUM-JIN YANG 1, LONGZHEN PIAO 1,2 , SANGHEE SHIN 1, SO-YEON SHIN 1, YUWEN LI 1,3 , HYUNJI LEE 1, QUANGDON TRAN 1, JISOO PARK 1, SUNTAEK HONG 4, DEREK P. BRAZIL 5, BRIAN A. HEMMINGS 6, SEON-HWAN KIM 7 and JONGSUN PARK 1

1Metabolic Syndrome and Cell Signaling Laboratory, Department of Pharmacology and Medical Science, Institute for Cancer Research, and 7Department of Neurosurgery, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon, Republic of Korea; 2Department of Oncology, Yanbian University Hospital, Yanji, P.R. China; 3Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi’an, P.R. China; 4Laboratory of Cancer Cell Biology, Department of , School of Medicine, Gachon University, Incheon, Republic of Korea; 5Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, U.K.; 6Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

Abstract. Unopposed phosphoinositide 3-kinase (PI3K) We demonstrated that reducing the level of 3- activity and 3-phosphoinositide production in Jurkat cells, phosphoinositides in Jurkat cells with pharmacological due to a mutation in the phosphatase and tensin homolog inhibitors of PI3K or expression of PTEN does not affect deleted on chromosome 10 (PTEN) tumor-suppressor PDK1 activity or its intracellular localization. We conclude, protein, results in deregulation of PH domain-containing therefore, that although Jurkat cells lack PTEN expression, proteins including the serine/threonine kinase PKB. In Jurkat only a subset of pathways downstream of PDK1 are cells, PKB is constitutively active and phosphorylated at the perturbed as a consequence of PTEN loss. activation-loop residue (Thr308). 3-Phosphoinositide- dependent protein kinase-1 (PDK1), an enzyme that also Phosphoinositide 3-kinase (PI3K) is a family of enzymes that contains a PH domain, catalyses Thr308 phosphorylation of play important roles in cellular proliferation, survival, PKB in addition to other kinase families such as PKC adhesion, cytoskeletal reorganization and motility (1, 2). isoforms. It is unknown, however, whether the loss of PTEN PI3K phosphorylates the 3’ position of the inositol ring of in Jurkat cells also results in unregulated PDK1 activity and inositol phospholipids, producing 3-phosphoinositides such whether such loss has an impact on activation-loop as phosphatidylinositol-(3,4)- bis phosphate (PtdIns (3,4)P 2) phosphorylation of other PDK1 substrates e.g. PKC. In this and phosphatidylinositol-(3,4,5)-triphosphate study, we addressed whether loss of PTEN in Jurkat cells (PtdIns(3,4,5)P 3). PtdIns(3,4)P 2 and PtdIns(3,4,5)P3 promote affects PDK1 catalytic activity and intracellular localization. membrane recruitment and activation of a number of proteins that contain pleckstrin homology domains (PH domains), including the serine/threonine kinase PKB (also known as Correspondence to: Dr. Seon-Hwan Kim, Department of AKT) (3, 4). Following PI3K activation, PKB translocates Neurosurgery, Institute for Cancer Research, College of Medicine, from the cytosol to the plasma membrane where it binds Chungnam National University, Daejeon, 35015, Republic of PtdIns(3,4)P 2/ PtdIns(3,4,5)P 3 via its N-terminal PH domain. Korea. Tel: +82 422807368, e-mail: [email protected] and Dr. The interaction of 3-phosphoinositides with the PH domain Jongsun Park, Department of Pharmacology and Medical Science, of PKB also induces a conformational change of the enzyme Metabolic Syndrome and Cell Signaling Laboratory, Institute for that facilitates phosphorylation of PKB on two key residues Cancer Research, College of Medicine, Chungnam National (5). These residues are Thr308, which is located on the University, Daejeon, 35015,Republic og Korea. Tel: +82 422806768, e-mail: [email protected] kinase domain and is known as the activation-loop site, and Ser473, that is located at the C-terminal hydrophobic-motif Key Words: PTEN, PI3K, PDK1, protein phosphorylation, Jurkat site. Thr308 phosphorylation is catalyzed by the upstream cells. enzyme 3-phosphoinositide-dependent protein kinase-1

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(PDK1) (5, 6). PDK1 also possesses a PH domain but the (18). Constitutive phosphorylation of PKC activation-loop in role of PtdIns(3,4)P2/PtdIns(3,4,5)P 3 in controlling the Jurkat cells might be attributed to lack of PTEN expression intracellular localization and/or activity of this enzyme is in this cell line. unresolved (5, 6). The mechanism of Ser473 phosphorylation In this study we addressed whether PTEN and PI3K on PKB is also not fully understood at present, with many activity in Jurkat cells affects PDK1 activity as well as its enzymes (including PKB itself) identified as the intracellular localization. hydrophobic-motif kinase(s) (7). The PI3K pathway is negatively regulated by the activity Materials and Methods of a lipid phosphatase called phosphatase and tensin Cell culture. The Jurkat E6.1 leukemia T-cell line and HuT-78 T-cell homolog deleted on chromosome 10 ( PTEN ), which lines were obtained from the American Type Culture Collection dephosphorylates the 3’ inositol ring of PtdIns(3,4)P 2 and (Manassas, VA, USA) and were cultured in RPMI-1640 containing PtdIns(3,4,5)P 3 (8, 9). PTEN is a tumor-suppressor gene 10% (v/v) heat-inactivated fetal calf serum (FCS), 50 U/ml located on chromosome 10q23, a region that suffers loss of penicillin, 50 μg/ml streptomycin and 2 mM L-glutamine (complete heterozygosity in many human cancer types (10, 11). medium) in a humidified chamber at 37˚C containing 5% CO 2. Furthermore, PTEN is deleted or mutated in a high PTEN- inducible tetracycline-inducible (Tet-on) Jurkat clones have previously been described (19) and were cultured in complete RPMI- percentage of cases of human glioblastomas and endometrial, 1640 medium with freshly added antibiotics G418 (100 μg/ml) and prostate, breast and hematopoietic cancer (10, 11). Many Hygromycin B (100 μg/ml) (Clontech, CA, USA). Three Tet-on leukemia T-cell lines, including the widely used Jurkat cell Jurkat clones were used in this study; clones 12 and 17 are PTEN - line, do not express functional PTEN protein due to inducible whilst clone 18 is a non- PTEN -expressing control clone. naturally-occurring mutations in both alleles of the PTEN The expression of PTEN was induced by the addition of 1 μg/ml gene (12). Tumors or cell lines that have lost expression of doxycycline (Clontech) to the cells for 48 h. To inhibit PI3K activity, PTEN have unopposed PI3K activity, resulting in elevated Jurkat cells were treated with 200 nM Wortmannin or 50 μM LY294002 for 3 h. Peripheral blood lymphocytes (PBLs) were basal levels of PtdIns(3,4)P /PtdIns(3,4,5)P . In PTEN -null 2 3 isolated from the blood of healthy volunteers on Lymphoprep buffy Jurkat cells, 50% of the total cellular pool of the PH domain- coats (Nycomed, Norway), washed twice with sterile phosphate- containing tyrosine kinase (interleukin-2-inducible T-cell buffered saline and incubated overnight in complete RPMI-1640 kinase; ITK) is localized in the plasma membrane-rich medium at 37˚C with 5% CO 2. Non-adherent cells were then fraction of unstimulated cells (13). harvested by centrifugation and processed for total cell lysates. Protein kinase C (PKC) is a family of serine/threonine Reagents and antibodies. PI3K inhibitor LY294002 was obtained kinases that are related to PKB due to homology in their from Alexis (Nottingham, UK). Wortmannin was obtained from respective kinase domain regions (14). However, PKCs do Sigma (St. Louis, MO, USA). Antibodies to the phosphorylated not possess PH domains and their activation is ultimately (pThr308 and pSer473) and total forms of PKB were obtained from controlled by γ ( PLC γ)- mediated Cell Signaling Technology (Beverly, MA, USA). Antibodies to diacylglycerol production, the formation of which recruits PTEN, extracellular signal-regulated kinase (ERK) 1/2 and PKC to membranes via their N-terminal C1 domains (15). horseradish peroxidase (HRP)-conjugated secondary antibodies Nonetheless, the PI3K pathway indirectly influences PKC were also obtained from Cell Signaling Technology. Antibody to lymphocyte function-associated antigen 1 (LFA-1) was obtained activity because PLC γ contains a PH domain and its activity from BD Biosciences (Oxford, UK). Antibodies to PKC β were and intracellular localization are regulated, in part, by the obtained from Seikagaku Corporation (Tokyo, Japan) and Zymed intracellular levels of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (San Francisco, CA, USA) and were used for immunoblotting and (16). Furthermore, PH domain-containing PDK1 immunoprecipitation respectively. Anti-PKC δ antibodies were phosphorylates the conserved activation-loop threonine obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) residue of PKCs (equivalent to Thr308 of PKB), thereby and BD Biosciences and were used for immunoblotting and potentially placing PKCs downstream of the PI3K pathway immunoprecipitation, respectively. Anti-PKC θ was obtained from BD Biosciences and used for both immunoprecipitation and (17). Because Jurkat cells lack PTEN and therefore have immunoblotting. Antibody that recognizes the phosphorylated elevated levels of PtdIns(3,4)P 2/PtdIns(3,4,5)P 3, it is possible activation-loop threonine of all PKC isoforms (P500 antibody) was that PTEN loss results in deregulation of the PDK1 and PLC generously provided by Alexandra Newton (University of γ pathways in this cell line, which in turn could be reflected California, San Diego, CA, USA) and was generated by Joanne in alteration of PKC activation-loop phosphorylation and Johnson in the Newton Laboratory. The specificity of this antibody intracellular localization. In agreement with this, It has been has been characterized elsewhere (20) and was used at a 1/1,000 reported that PKCs are phosphorylated at the activation-loop dilution. Sheep anti-human PDK1 antiserum was kindly donated by Dr. Dario Alessi (University of Dundee, Scotland, UK). HRP- residue in Jurkat cells and that, similarly to PKB labelled anti-sheep secondary antibody was from Sigma. A second phosphorylation, short-term cellular stimulation with antibody to PDK1, used to immunoprecipitate endogenous PDK1 antibodies to T-cell receptor (TCR)/cluster of differentiation for kinase assays, was obtained from Upstate Biotech 28 (CD28) does not affect the level of PKC phosphorylation (Charlottesville, VA, USA).

5416 Yang et al : PDK1 Activity in Jurkat T-Cells

Cell lysis, immunoprecipitation and cell fractionation. Total cell lysates, immunoprecipitation and subcellular fractionation were performed as described previously (21).

SDS-PAGE and western blotting. The western blot analysis was performed as described previously (22, 23). Total lysates, immunoprecipitates and fractionated lysates were resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) gels, transferred to polyvinylidene fluoride (PVDF) membranes by the semi-dry technique and probed with the relevant antibodies according to the manufacturers’ instructions. Immunoreactive bands were visualised with the Phototope-HRP detection system (Cell Signaling) and subsequent exposure to Kodak light-sensitive film (Cedex, France). Where indicated, the PVDF membrane was stripped of its antibodies by incubation in stripping buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS and 100 mM β- mercaptoethanol] for 30 min at 65˚C, followed by washing, blocking and reprobing with antibodies as outlined above.

PDK1 activity assays. Assays for PDK1 activity were performed as outlined in (21) using 100 μM Suntide (RRKDGATMKTFCGTPE) Figure 1. Lack of phosphatase and tensin homolog deleted on as substrate, except that endogenous PDK1 was immunoprecipitated chromosome 10 (PTEN) phosphatase expression in Jurkat cells in with a commercially available antibody to PDK1 (Upstate Biotech) comparison to peripheral blood lymphocytes (PBLs) and Hut-78 T-cells. and assayed under specific conditions. A: Unstimulated Jurkat cells, and HuT-78 T-cell lysates were probed for the expression of PTEN by western blotting. B: The same lysates were probed for phosphorylated (pThr308) and total protein kinase B (PKB). Results

Inhibitors of PI3K, and expression of PTEN do not affect the catalytic activity nor intracellular localization of endogenous PDK1 in Jurkat cells. Jurkat cells lack expression of the lipid phosphatase PTEN, an effect that results in unopposed PI3K of both enzymes, PDK1, could be unregulated in these cells activity and therefore high basal levels of PtdIns(3,4)P 2 and due to PTEN loss and unopposed PI3K activity. PtdIns(3,4,5)P 3 in these cells (13, 24). Unopposed PI3K To investigate whether unopposed PI3K activity results in activity results in constitutive activation and/or membrane deregulation of PDK1 activity and intracellular localization localization of a number of kinases that are dependent on in Jurkat cells, the cells were incubated with PtdIns(3,4)P 2/PtdIns(3,4,5)P 3 for activity, such as PKB. pharmacological inhibitors of PI3K (wortmannin and Preliminary experiments also confirmed that our Jurkat cells LY294002) to reduce the levels of 3-phosphoinositides and grown in culture lacked any detectable expression of PTEN the catalytic activity and subcellular localization of (Figure 1A). Although the expression of this phosphatase endogenous PDK1 was analyzed. As shown in Figure 2A, was absent from the Jurkat cell line, PTEN was readily endogenous PDK1 displayed high basal activity towards a detectable in lysates derived from unstimulated PBLs. In peptide substrate based on the activation-loop sequence of agreement with other reports, we found that PTEN was also PKB when isolated from unstimulated Jurkat cells. Treating expressed in the HuT-78 lymphoma T-cell line (25, 26). To Jurkat cells with 50 μM LY294002 for 30 min 3 h did not confirm that 3-phosphoinositides such as PtdIns(3,4,5)P 3 are affect the catalytic activity or expression level of PDK1. abnormally high in the Jurkat cell line, the phosphorylation PTEN was expressed in Jurkat cells under the control of status of PKB at the activation-loop residue (Thr308) was a Tet-on expression system; addition of doxycycline to the determined by immunoblotting (the phosphorylation state of cell culture medium of these cells induces the expression of PKB at Thr308 and Ser473 has been shown to accurately PTEN. For this study, three PTEN -inducible Tet-on Jurkat reflect the levels of 3-phosphoinositides in this cell line) (13, clones were used; clone 12 and clone 17 can be induced to 24). Immunoblotting of Jurkat lysates with a express PTEN by the addition of doxycycline, whilst clone phosphorylation-specific antibody to PKB (pThr308) 18 is a negative control clone that does not express PTEN determined that Jurkat cells, in contrast to normal PBLs and under the same induction conditions. As shown in Figure 2B, unstimulated HuT-78 T-cells, contained phosphorylated PKB addition of doxycycline to the culture medium of Jurkat Tet- (Figure 1B). PKB was expressed at a comparable level in all on clones 12 and 17 for 48 hours resulted in the up- cells analyzed. Non-inducible phosphorylation of both PKC regulation of PTEN in these cells (but not in clone 18). The (18) and PKB in Jurkat cells implies that the upstream kinase expression of PTEN in Jurkat Tet-on cells did not

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Figure 2. Inhibitors of phosphoinositide 3-kinase (PI3K), and expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) do not affect the catalytic or intracellular localization of endogenous 3-phosphoinositide-dependent protein kinase-1 (PDK1) in Jurkat cells. A: Jurkat cells were treated with 50 μM PI3K inhibitor LY294002 (+) or control (−) for 30 min and 3 h. Lysates were immunoprecipitated with an antibody to PDK1 and immunoprecipitates were tested for PDK1 activity in an in-vitro kinase assay. An aliquot of each lysate was analyzed by western blotting for the expression of PDK1 or actin as a loading control. B: PTEN-inducible Tet-on Jurkat clones (indicated with #) were left untreated or treated with doxycycline (Dox) for 48 h to induce expression of PTEN. Clones 12 and 17 are PTEN-inducible, while clone 18 is a PTEN-negative control. Lysates were prepared and were immunoblotted for expression of PTEN, PDK1 or actin as a loading control. The same lysates were immunoprecipitated with an antibody to PDK1 and immunoprecipitates were tested for PDK1 activity in an in-vitro kinase assay. C: Wild-type Jurkat cells were treated with 50 μM LY294002 for up to 6 hours or with control (−), cells were fractionated into cytosol- and membrane-rich fractions and probed by western blotting for PDK1. C: Cytosol-rich fraction, M: membrane-rich fraction.

significantly affect endogenous PDK1 activity (Figure 2B). to phosphorylate its downstream substrates in Jurkat cells, we Furthermore, the expression of PTEN did not affect the analyzed the activation-loop phosphorylation status of PKC expression of PDK1. The effect of PI3K inhibitors on the isoforms βI, δ and θ following PI3K inhibition with /without intracellular localization of endogenous PDK1 in Jurkat cells PTEN expression. As shown in Figure 3A, treating Jurkat was also determined by subcellular fractionation and cells with wortmannin or LY294002 did not affect the immunoblotting. Treating Jurkat cells with LY294002 for up phosphorylation status of PKC isoforms at the corresponding to 6 hours did not affect the proportion of PDK1 associated activation-loop threonine site. Incubation of Jurkat cells with with the membrane-rich fraction (Figure 2C). These LY294002 for longer periods (up to and including 24 h) also experiments conclusively show that although PDK1 contains had no effect on the phosphorylation status of PKC isoforms a PH domain, alterations in the levels of 3-phosphoinositides in Jurkat cells (data not shown). The phosphorylation status in Jurkat cells do not influence its function. of PKC θ at the activation-loop site (Thr538) was confirmed using a commercially available phospho-specific antibody Inhibitors of PI3K, and expression of PTEN do not affect following PI3K inhibition; inhibitors of PI3K did not PKC activation-loop phosphorylation nor intracellular modulate the phospho-status of PKC θ at Thr538 in Jurkat localization in Jurkat cells. To confirm that reducing the level cells (data not shown). The effect of PTEN expression on the of 3-phosphoinositides does not affect the capacity of PDK1 phosphorylation status of PKC isoforms at the activation-loop

5418 Yang et al : PDK1 Activity in Jurkat T-Cells

Figure 3. Inhibitors of phosphoinositide 3-kinase (PI3K), and expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) do not affect protein kinase C (PKC) activation-loop phosphorylation or intracellular localization in Jurkat cells. A: Jurkat cells were treated for 3 h with 200 nM wortmannin (W), 50 μM LY294002 (LY) or vehicle control (−) and lysates were immunoprecipitated with antibodies to PKC βI, δ and θ. Immunoprecipitates were probed by western blotting with an antibody that recognizes the phosphorylated activation-loop threonine of PKC isoforms. As a relative loading control, the blots were stripped and reprobed for total PKCs. B: PTEN-inducible Tet-on Jurkat clones were left untreated or treated with doxycycline (Dox) for 48 h to induce PTEN expression and lysates were immunoprecipitated with antibodies to PKC βI, δ and θ, or a non-specific monoclonal antibody. Immunoprecipitates were probed by western blotting with an antibody that recognizes the phosphorylated activation-loop threonine of PKC isoforms. As a relative loading control, the blots were stripped and reprobed for total PKCs.

residue in Jurkat cells was also analyzed. PTEN expression group (18), PKC βI was recovered predominantly from the did not affect the phosphorylation status of PKC isoforms cytosol-rich fraction of unstimulated Jurkat cells with PKC βI, δ and θ ( Figure 3B ). The phosphorylation status of minimal amounts present in the membrane-rich fraction PKC θ following PTEN expression was also confirmed in (Figure 4A). Treating the cells with LY294002 for 3 h did Jurkat cells using a commercially available phospho-specific not affect the intracellular localization of this kinase. In (Thr538) activation-loop antibody. The expression of PTEN contrast to PKC βI, unstimulated Jurkat cells contain higher did not modulate the phospho-Thr538 status of PKC θ ( data levels of membrane-localized PKC δ and θI, but treating the not shown ). cells with LY294002 did not affect the intracellular We next investigated the effect of PI3K inhibitors and distribution of these kinases (Figure 4A). The effect of PTEN expression of PTEN on the subcellular localization of PKC expression on the subcellular localization of PKC isoforms isoforms in Jurkat cells. As previously published by other was also investigated. As shown in Figure 4B, PTEN

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Figure 4. Inhibitors of phosphoinositide 3-kinase (PI3K), and expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in Jurkat cells do not affect the intracellular localization of protein kinase C (PKC) isoforms. A: Jurkat cells were treated with 50 μM LY294002 (LY) or control (−) for 3 h and the cells were fractionated into cytosol- and membrane-rich fractions. The fractions were probed by western blotting for total PKC βI, δ and θ. B: PTEN-inducible Tet-on Jurkat clones were left untreated or treated with doxycycline (Dox) for 48 h to induce expression of PTEN. The cells were fractionated into cytosol- and membrane-rich fractions and probed by western blotting for total PKC βI, δ and θ. C: Cytosol-rich fraction; M: membrane-rich fraction.

expression did not affect the intracellular distribution of activation-loop residue of many other kinase family members PKC βI, δ and θ isoforms. Furthermore, PTEN expression (equivalent to Thr308 of PKB), including PKC isoforms, p70 did not alter the anti-TCR/CD28-induced translocation of ribosomal S6 kinase (S6K), p90 ribosomal S6 kinase (RSK) these PKC isoforms to the detergent-soluble membrane and serum and glucocorticoid kinases (SGK) (5, 6). Although fraction (data not shown). We conclude, therefore, that PDK1 PDK1 contains a PH domain, the role of 3-phosphoinositides and PKC signaling is not perturbed as a consequence of in controlling PDK1 localization and activity is poorly PTEN loss in Jurkat cells. understood. For example, it has been reported that cellular stimulation and PI3K activation modulates PDK1 activity Discussion and localization (28-32), whilst other studies have shown that PDK1 is a constitutively active enzyme and functions Proteins that contain PH domains are abnormally regulated independently of the PI3K pathway (33-35). Nevertheless, in Jurkat cells due to mutations in both alleles of PTEN . For because PDK1 contains a PH domain, its intracellular example, the PH domain-containing enzyme PKB is localization or activity is potentially unregulated in Jurkat constitutively activated and phosphorylated at the activation- cells as a consequence of PTEN loss. loop residue (Thr308) (13, 27). The enzyme PDK1 catalyses In this study we sought to determine whether unregulated Thr308 phosphorylation on PKB. PDK1 is known as a PI3K activity in Jurkat cells influences PDK1 activity and master kinase since this enzyme also phosphorylates the intracellular localization. We found that inhibition of PI3K

5420 Yang et al : PDK1 Activity in Jurkat T-Cells activity or expression of PTEN in Jurkat cells reduced the then it could be surmised that all of the cellular pool of high basal levels of 3-phosphoinositides but these treatments PDK1 would be localized in the membrane fraction of in parallel did not affect the catalytic activity or intracellular untreated Jurkat cells. This is not the case, as we found that localization of endogenous PDK1. The high basal activity of PDK1 was also localized to the cytosol of unstimulated PDK1 in quiescent, PTEN-expressing cells (or Jurkat cells Jurkat cells. Furthermore, if 3-phosphoinositides were with low levels of 3-phosphoinositides) may be explained primarily responsible for PDK1 membrane localization, then perhaps by a report that the enzyme phosphorylates its own reducing the levels of 3-phosphoinositides by chronically activation-loop residue Ser241 (analogous to Thr308 of treating cells with PI3K inhibitors for extended time periods PKB) and that this site is constitutively phosphorylated in would be expected to abolish membrane localization. Both unstimulated cells (36). It should be noted that although we and Alessi and colleagues (33) have demonstrated that PDK1 exhibits high basal activity in T-cells per se , chronically treating Jurkat and HEK-293 cells, respectively, mechanisms exist to further increase its activity in other cell with LY294002 does not abolish membrane localization of types in vivo , including phosphorylation on tyrosine, serine PDK1; these experiments suggest entities other than 3- and threonine residues (6). It is also known that sphingosine, phosphoinositides may regulate PDK1 membrane an intracellular lipid with potent biological activity, has been localization. A number of scaffolding proteins and signaling reported to directly increase autophosphorylation of PDK1 molecules that associate with PDK1 have recently been (37). Whether these factors influence PDK1 activity in T- identified that could explain how the enzyme is both lymphocytes remains to be investigated however. cytosolic and tethers to the plasma membrane. For example, That reducing the levels of 3-phosphoinositides in Jurkat the adaptor protein, growth factor receptor-bound protein 14 cells does not perturb the intracellular localization of a PH (GRB14) binds PDK1 and regulates its membrane domain-containing enzyme such as PDK1 may be explained localization in HEK-293, MCF-7 and HeLa cells; this by the fact that although PDK1 has been conclusively interaction between PDK1 and GRB14 is important for PKB shown to bind to PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 in vitro (but not PKC) phosphorylation (37). It has been reported that and in vivo , the affinity of the PH domain of PDK1 for 3- PDK1 can also bind to PtdIns(4,5)P 2, the substrate of PI3K, phosphoinositides is 20-fold higher than that of the PH albeit with a 15-fold lower affinity than 3-phosphoinositides domain of PKB (33, 38, 39). Furthermore, it appears that in insulin-responsive cells (33). This interaction may be the PH domain of PDK1, like PKB, serves to regulate the significant in localizing PDK1 to the plasma membrane enzyme; engagement of this module is thought to relieve independently of 3-phosphoinositides in vivo , as basal levels auto-inhibition (30, 40). Because of its high affinity for 3- of PtdIns(4,5)P 2 are known to be up to 200-fold higher than phosphoinositides, it is likely that PDK1 is capable of the basal levels of 3-phosphoinositides in these cells (33). binding to very low basal levels of PtdIns(3,4)P 2/ PDK1 is also known to localize to the cytosol in all cell PtdIns(3,4,5)P 3, which serve to maintain the enzyme at the types studied to date. Indeed, PDK1 phosphorylates the plasma membrane in a high activity form in T lymphocytes activation-loops of many cytosolic substrates such as S6K (i.e. phosphorylated on its own activation-loop, relieved of and RSK, enzymes that do not contain plasma membrane- auto-inhibition and capable of substrate phosphorylation). binding modules (5, 6). Recent evidence suggests that In contrast, these low levels of 3-phosphoinositides would inositol phospholipids such as Ins(1,3,4,5,6)P 5 and IP6 are be below the threshold concentration required to sustain important for localizing PDK1 to the cytosol (41). Whether PKB plasma membrane localization and as such PKB would these scaffolding proteins and lipids control PDK1 accumulate in a non-phosphorylated form in the cytosol. In intracellular localization in T-lymphocytes has not been support of this theory, we found that reducing the explored. We cannot rule-out, however, that cytosolic PDK1 intracellular levels of 3-phosphoinositides did not modulate in Jurkat cells, similarly to cytosolic PKB, represents active the activity or subcellular localization of endogenous PDK1, enzyme in a membrane to nucleus transition; indeed PDK1 while the same treatments abolished the distribution of PKB is known to localize to the nucleus in other cell types in a in the membrane fraction and led to kinase inactivation PI3K-dependent manner (42, 43). (dephosphorylated at Thr308/Ser473). Therefore, although We have also shown that the subcellular localization of its name points to a dependency on 3-phosphoinositides for PKC isoforms are not affected by changes in the levels of 3- function, in effect these lipids do not modulate the intrinsic phosphoinositides in Jurkat cells. Interestingly, it has been activity or subcellular localization of PDK1 in T demonstrated that proliferating leukemic Jurkat cells, in lymphocytes. contrast to their normal T-cell counterparts, contain high It is possible that entities other than 3-phosphoinositides levels of plasma membrane-associated PKC (44) (Figure 4). could maintain PDK1 in a constitutively active state in the High levels of PKC in the membrane fraction of plasma membrane of T-lymphocytes; if 3-phosphoinositides unstimulated Jurkat cells could potentially be attributed to were primarily responsible for PDK1 membrane localization, PTEN loss. However, we have found that inhibiting PI3K or

5421 ANTICANCER RESEARCH 37 : 5415-5423 (2017) expressing PTEN does not abrogate the association of PKC 5 Mora A, Komander D, van Aalten DM and Alessi DR: PDK1, with the membrane-rich fraction (Figure 4). Furthermore, the the master regulator of AGC kinase . Semin PTEN-expressing HuT-78 T-cell line also contains high Cell Dev Biol 15 : 161-170, 2004. 6 Storz P and Toker A: 3’-phosphoinositide-dependent kinase-1 levels of PKC in the membrane fraction (data not shown). (PDK-1) in PI 3-kinase signaling. Front Biosci 7: d886-902, 2002. That reducing 3-phosphoinositide levels in Jurkat cells does 7 Dong LQ and Liu F: PDK2: the missing piece in the receptor not affect PKC intracellular localization implies that PLC tyrosine kinase signaling pathway puzzle. Am J Physiol activity is unaltered as a consequence of PTEN loss in this Endocrinol Metab 289 : E187-196, 2005. cell line. How is this so, if PLC contains a PH domain? This 8 Besson A, Robbins SM and Yong VW: PTEN/MMAC1/TEP1 in is because most signaling events in T-lymphocytes require a signal transduction and tumorigenesis. Eur J Biochem/FEBS secondary signal of some sort in addition to PI3K activation. 263 : 605-611, 1999. For example, although 50% of total cellular pool of Itk is 9 Cantley LC and Neel BG: New insights into tumor suppression: PTEN suppresses tumor formation by restraining the membrane-bound in Jurkats (26), the enzyme is not active phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci because catalytic activation of Itk also requires ZAP-70- USA 96 : 4240-4245, 1999. dependent activation-loop phosphorylation (ZAP-70) is only 10 Bendib M, Badescu A and Handra-Luca A: PTEN expression in activated upon TCR ligation). Similarly, although PLC also colorectal adenomas: relationship to morphology and cell contains a PH domain, the enzyme is catalytically inactive heterogeneity. Pathol Res Pract 211 : 248-251, 2015. since this enzyme also requires ZAP-70-dependent 11 Yoshimoto M, Ludkovski O, DeGrace D, Williams JL, Evans A, phosphorylation on Tyr783 for optimal activity. In contrast, Sircar K, Bismar TA, Nuin P and Squire JA: PTEN genomic deletions that characterize aggressive prostate cancer originate other PH domain-containing enzymes, such as PDK1, are close to segmental duplications. Gene Chromosome Cancer 51 : also not influenced by the level of 3-phosphoinositides in 149-160, 2012. Jurkat cells at all. It is interesting to note, therefore, that 12 Tesio M, Trinquand A, Macintyre E and Asnafi V: Oncogenic although Jurkat cells lack PTEN, only a subset of PI3K- PTEN functions and models in T-cell malignancies. Oncogene dependent pathways are perturbed as a consequence of the 35(30) : 3887-3896, 2016. absence of this tumor-suppressor protein (such as PKB since 13 Shan X, Czar MJ, Bunnell SC, Liu P, Liu Y, Schwartzberg PL and the activation of this kinase is solely dependent on PI3K Wange RL: Deficiency of PTEN in Jurkat T-cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness activity). In summary, we have demonstrated that to CD3 stimulation. Mol Cell Biol 20 : 6945-6957, 2000. unregulated PI3K activity does not affect the catalytic 14 Antal CE and Newton AC: Tuning the signalling output of activity or intracellular localization of PDK1. To the best of protein kinase C. Biochem Soci Trans 42 : 1477-1483, 2014. our knowledge, this is the first study to directly address the 15 Zeng L, Webster SV and Newton PM: The biology of protein role of the PI3K/PTEN pathway on endogenous PDK1 kinase C. Adv Exp Med Biol 740 : 639-661, 2012. function. 16 Falasca M, Logan SK, Lehto VP, Baccante G, Lemmon MA and Schlessinger J: Activation of phospholipase C gamma by PI 3- Acknowledgements kinase-induced PH domain-mediated membrane targeting. EMBO J 17 : 414-422, 1998. 17 Newton AC: Regulation of the ABC kinases by phosphorylation: This work was financially supported by research fund of Chungnam protein kinase C as a paradigm.Biochem J 370: 361-371, 2003. National University in 2015 (Jongsun Park) and by the National 18 Freeley M, Volkov Y, Kelleher D and Long A: Stimulus-induced Research Foundation of Korea (NRF) grant funded by the Korea phosphorylation of PKC theta at the C-terminal hydrophobic- Government (MEST) (NRF-2012M3A9B6055302, NRF- motif in human T-lymphocytes. 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