Salivary glands are innervated by sympathetic and parasympathetic neurons, which release neurotransmitters that promote fluid secretion and exocytosis when they bind to muscarinic and ␤-adrenergic receptors, respectively. Signaling pathways downstream of these receptors are mainly distinct, but there is cross-talk that affects receptor-dependent events. Here we report that the ␤-adrenergic ligand isoproterenol blocks increases in extracellular signal-related kinase (ERK) phosphorylation, a protein kinase C-dependent event promoted by the muscarinic receptor ligand carbachol in freshly dispersed rat parotid acinar cells. The inhibitory action of isoproterenol was reproduced by cAMP stimuli ( Almost all mammalian cells have multiple types of heptahelical G-protein-coupled receptors. The muscarinic receptor and ␤-adrenergic receptor are G-protein-coupled receptors that affect the physiological activities of many cells, and neurotransmitters binding to these receptors produce well defined and mainly separate functional effects in salivary glands, which are innervated by both sympathetic and parasympathetic neurons (1). Parotid acinar cells express M3 muscarinic receptors (2), which are coupled via G q to phospholipase C, and acetylcholine and M3R 2 ligands binding to this receptor initiate the production of the second messengers inositol trisphosphate (InsP 3 ) and diacylglycerol, which elevate the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and activate PKC. The increase in intracellular Ca 2ϩ initiates the opening of ion channels, electrolyte and fluid secretion, and subsequently, the formation of saliva in the oral cavity (3). In contrast, the activation of parotid ␤-adrenergic receptors promotes exocytosis and the release of amylase protein into the oral cavity. ␤-Adrenergic receptors are coupled via G s to adenylyl cyclase, and receptor stimulation generates the production of cAMP (cyclic adenosine monophosphate) and the activation of cAMP-dependent protein kinase (protein kinase A (PKA)) and a signaling cascade downstream of PKA. Muscarinic receptors also can promote exocytosis, but this is more limited than that due to ␤-adrenergic signaling (4). In many cells cAMP also can activate exchange proteins directly activated by cAMP (Epac), which are guanine nucleotide exchange factors for the GTPase Rap. Epac is a cAMP-dependent PKA-independent protein that can mediate various signaling and functional events in salivary glands and other cells (5-7). This can produce complications in evaluating the mechanisms of cross-talk that exist when there is combined muscarinic and ␤-adrenergic signaling, which is a focus of the present study.Isoproterenol has other effects on parotid acinar cells in addition to promoting exocytosis of secretory granules. It

Interleukin-3 (IL-3) mediates hematopoietic cell survival and proliferation via several signaling pathways such as the Janus kinase/signal transducer and activator of transcription pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphoinositide-3 kinase (PI-3K) pathway. Mammalian target of rapamycin (mTOR) is one of the downstream targets of the PI-3K pathway, and it plays an important role in hematopoiesis and immune cell function. To better elucidate how mTOR mediates proliferation signals from IL-3, we assessed the role of S6 kinase 2 (S6K2), one of the downstream targets of mTOR, in IL-3 signaling. We show that S6K2 is activated by IL-3 in the IL-3-dependent Ba/F3 cell line and that this is mediated by mTOR and its upstream activator PI-3K but not by the MAPK kinase/extracellular signal-regulated kinase pathway. S6K2 is also activated in primary mouse bone marrow-derived mast cells upon IL-3 stimulation. Expression of a rapamycin-resistant form of S6K2, T388E, in Ba/F3 cells provides a proliferation advantage in the absence or presence of rapamycin, indicating that S6K2 can potentiate IL-3-mediated mitogenic signals. In cells expressing T388E, rapamycin still reduces proliferation at all doses of rapamycin, showing that mTOR targets other than S6K2 play an important role in IL-3-dependent proliferation. Cell-cycle analysis shows that T388E-expressing Ba/F3 cells enter S phase earlier than the control cells, indicating that the proliferation advantage may be mediated by a shortened G1 phase. This is the first indication that S6K2 plays a role in IL-3-dependent cell proliferation.

Soltoff SP, Hedden L. Regulation of ERK1/2 by ouabain and Na-KATPase-dependent energy utilization and AMPK activation in parotid acinar cells. Am J Physiol Cell Physiol 295: C590 -C599, 2008. First published July 16, 2008 doi:10.1152/ajpcell.00140.2008.-We previously found that the phosphorylation of ERK1/2 by submaximal concentrations of the muscarinic receptor ligand carbachol was potentiated in rat parotid acinar cells exposed to ouabain, a cardiac glycoside that inhibits the Na-K-ATPase. We now report that this signaling phenomenon involves the prevention of negative regulation of extracellular signalregulated kinase-1/2 (ERK1/2) that is normally mediated by AMPactivated protein kinase (AMPK). Carbachol increases the turnover of the ATP-consuming Na-K-ATPase, reducing intracellular ATP and promoting the phosphorylation/activation of the energy sensor AMPK. Ouabain blocks the reduction in ATP and subsequent AMPK phosphorylation, which is regulated by the AMP-to-ATP ratio. The ouabain-promoted enhancement of ERK1/2 phosphorylation was not reproduced in Par-C10 cells, an immortalized rat parotid cell line that did not respond to carbachol with an ATP reduction and that employs an upstream AMPK kinase (Ca 2ϩ /calmodulin-dependent protein kinase kinase, CaMKK) different from that (LKB1) in native cells. In native parotid cells, inhibitory effects of AMPK on ERK1/2 signaling were examined by activating AMPK with 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR), which is converted to an AMP mimetic but does not alter parotid ATP levels. AICAR-treated cells display increases in AMPK phosphorylation and a reduced phosphorylation of ERK1/2 subsequent to activation of muscarinic and P2X7 receptors, which promote increases in Na-K-ATPase turnover, but not upon epidermal growth factor receptor activation. These results suggest that carbachol-initiated AMPK activation can produce a negative feedback on ERK1/2 signaling in response to submaximal muscarinic receptor activation and that increases in fluid secretion can modulate receptor-initiated signaling events indirectly by producing ion transport-dependent decreases in ATP. adenosine 5Ј-triphosphatase; calmodulin-dependent protein kinase

The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase ␣ subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the ␣1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified The Na,K-ATPase, or sodium pump, is an important ion transport protein that maintains the electrochemical gradient across the plasma membrane of eukaryotic cells. It is an integral plasma membrane protein that transports three Na ϩ ions for every two K ϩ ions, an event that consumes one molecule of ATP. As such, it participates in fluid and electrolyte secretion and cell volume regulation and is part of a network of ion transporters that regulate cellular ionic changes during basal, stimulated, and pathological conditions. The basic functional unit of the Na,K-ATPase protein consists of an ␣ subunit responsible for the catalytic activity, a glycosylated ␤ subunit, and in some cells, an FXYD protein. There are multiple isoforms of ␣ and ␤ subunits (1) and different FXYD proteins (2).Although it has been known for decades that the Na,KATPase activity is regulated by the intracellular and extracellular ionic composition, regulation can also occur by the phosphorylation of the ␣ subunit by various kinases (for review, see Ref.3). The ␤ subunit and FXYD proteins may also play regulatory roles under some conditions. Various studies demonstrated that Ser 943 , a site on the ␣ subunit C terminus, was a target for PKA phosphorylation, and Ser 16 and Ser 23 on the N terminus were identified as PKC phosphorylation sites (4 -7). In addition to contrasting results obtained from different species and differences between in vitro and intact cells (below), investigators have used different numbering of the ␣ subunit amino acids because the first five are cleaved during biosynthesis and production of the mature protein. There are conflicting studies concerning the effects of different kinases on the ␣ subunit phosphorylation and Na,KATPase activity. The phosphorylation of the ␣ subunit by PKC on Ser 23 and unspecified sites stimulated the Na,K-ATPase activity in intact cells (9, 10), in some cases due to its enhanced insertion into the plasma membrane (11). Alternatively, the PKC-mediated phosphorylation of ␣ produced a reduction in Na,K-ATPase activity due to its endocytosis and internalization (12)(13)(14). In some studies, the ␣ subunit was preferentially phosphorylated by members of the classical PKC family (cPKC: 2 ␣, ␤, ␥) when compared with novel PKC family members (nPKC: ␦, ⑀, ) (11, 15). However, both cPKC (PKC␤I) and nPKC (PKC␦) proteins were reported to regulate ␣ subunit phosphorylation (16), and PKC, an atypical PKC family member, also phosphorylated the ␣ subunit on Ser 23 (13). In addition to PKC, other kinases were reported to phosphorylate the ␣ s...

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