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 Biochemistry, 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 5415 ANTICANCER RESEARCH 37 : 5415-5423 (2017) (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 phospholipase C 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.