Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

No abstract

No abstract

Regulation of the activity of the extracellular signal regulated kinase (ERK) mitogen-activated protein kinases was examined in Rat-1 HIR, a fibroblast cell line overexpressing the human insulin receptor. Insulin or phorbol ester induced partial activations of ERKs, while a combination of insulin and phorbol ester resulted in a synergistic activation. Preincubation with phorbol ester increased the subsequent response to insulin. Phorbol ester did not enhance tyrosine phosphorylation of the insulin receptor. Insulin did not enhance activation of phospholipase D in response to phorbol ester. Lysophosphatidic acid also acted synergistically with insulin to induce ERK activation. Lysophosphatidic acid alone had little effect on ERK, and did not activate phospholipase D. The combination of phorbol ester and insulin maintained tyrosine phosphorylation of focal adhesion kinase, while insulin alone decreased its tyrosine phosphorylation. Phorbol ester induced phosphorylation of Shc on serine/threonine, while insulin induced tyrosine phosphorylation of Shc and Shc-Grb2 binding. These results suggest that full activation of ERKs in fibroblasts can require the cooperation of at least two signaling pathways, one of which may result from a protein kinase C-dependent phosphorylation of effectors regulating ERK activation. In this manner, phorbol esters may enhance mitogenic signals initiated by growth factor receptors.The ERK 1 mitogen-activated protein kinases (MAPKs), which are activated in response to a wide variety of growth factors, hormones, and other mitogenic stimuli, have been linked to the induction of cell proliferation (1). The insulin receptor, a tyrosine kinase, can elicit both metabolic and mitogenic responses (2). The insulin receptor binds to several effectors, with binding to insulin receptor substrate-1 (IRS-1) mediating most of the metabolic effects of insulin. Mutation or deletion of some of the phosphorylatable tyrosines in the insulin receptor can enhance its mitogenic activity (3), suggesting that multifunctionality of the receptor may compromise its mitogenic potential. Phorbol ester tumor promoters can act additively or synergistically with insulin to induce mitogenesis (4, 5).The mitogenic activity of the insulin receptor has been correlated with its ability to activate MAPK, a response that is mediated predominantly through interaction of the insulin receptor with Shc (6). IRS-1 and Shc compete for binding to Grb2, such that IRS-1 may either inhibit (7) or enhance (8) activation of MAPK. Tyrosine-phosphorylated Shc binds to the adapter Grb2, causing disassembly of SOS from Grb2 resulting in GDP-GTP exchange on Ras (9). GTP-bound Ras promotes activation of Raf-1 kinase (1, 10). Raf-1 phosphorylates and activates MEK (mitogen-activated protein kinase kinase/ERK kinase), which phosphorylates and activates MAPK. One downstream effector of MAPK is RSK, another protein serine/threonine kinase. It is clear that the insulin receptor initiates signals that are independent of MAPK activation (reviewed in Ref...

PC12 neuronal cells express a membrane phospholipase D (PLD) activity that is detected at similar levels in undifferentiated or differentiated cells. The regulation of this activity by agonists was explored. Membrane phospholipase D activity was increased by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or with nerve growth factor. The ability of PMA to activate PLD was confirmed in intact PC12 cells. Basal activity of PLD in membranes was reduced in RG20, a PC12 cell line overexpressing the human ␣ 2A -adrenergic receptor. PMA did not increase PLD activity in RG20 cells, as assessed both in membrane preparations and in intact cells. Cyclic AMP levels did not regulate phospholipase D activity in either cell type. However, incubation of RG20 cells with the ␣ 2 -adrenergic antagonist rauwolscine or with pertussis toxin increased membrane PLD activity and restored activation of PLD by PMA. These data suggest that the effects of the overexpressed ␣ 2A -adrenergic receptor on PLD activity are mediated by precoupling of the receptor to the heterotrimeric GTP-binding protein, G i , but are independent of adenylate cyclase regulation. The results of this study suggest that membrane phospholipase D activity can be negatively regulated via G i in PC12 cells. Phospholipase D (PLD)1 isoforms have been sequenced from yeast (1-3) and mammalian (4) cells. It appears that more than one form of PLD is expressed in mammals (5-8). PLDs can be activated in response to extracellular signals such as growth factors, hormones, and neurotransmitters (9) and by phorbol esters that stimulate protein kinase C (10). Hydrolysis of phosphatidylcholine (PC) by PLD produces phosphatidic acid (PA). PA is proposed to play a role in signal transduction as a lipid mediator or mediator precursor (11)(12)(13)(14). Some PLDs are regulated by the small GTP-binding proteins . However, the full range of effectors and mediators regulating different isoforms of PLD remains to be elucidated. This laboratory has characterized regulated PLDs in yeast and mammalian cells (10, 18 -20). In this study, we explore the role of a heterotrimeric GTP-binding protein, G i , in the regulation of PLD activity in a neuronal cell line.Signal transduction pathways have been extensively studied in PC12, a rat phaeochromocytoma cell line that can be induced to differentiate to a neuronal phenotype. PC12 transfected with the ␣ 2A -adrenergic receptor (␣ 2A AR) have been used to examine coupling of this receptor to its effectors (21). The ␣ 2A AR couples to the heterotrimeric GTP-binding protein G i , is widely expressed, and mediates the central hypotensive effects of ␣ 2 agonists (22,23). G i proteins are heterotrimeric GTP-binding proteins containing an ␣ i subunit. They are coupled to inhibition of adenylate cyclase as well as to pathways involving additional effectors, such as small GTP-binding proteins (24). In this study, ␣ 2A AR-expressing PC12 cells were used to examine the potential role of G i in regulating PLD activity. MATERIALS AN...