LETTER TO NEUROSCIENCE SEROTONIN PERSISTENTLY ACTIVATES THE EXTRACELLULAR SIGNAL-RELATED KINASE IN SENSORY NEURONS OF APLYSIA INDEPENDENTLY OF cAMP OR PROTEIN KINASE C J. R. DYER, a F. MANSEAU, b V. F. CASTELLUCCI b AND W. S. SOSSIN a * a Department of Neurology and Neurosurgery, McGill University, Mon- treal Neurological Institute, Room 776, 3801 rue University, Montreal, Que., Canada H3A 2B4 b Laboratoire de Neurobiologie et comportement, Centre de Recherche en Sciences Neurologiques, De ´ partement de physiologie, Universite ´ de Montre ´ al, Montreal, Que., Canada Abstract—Activation of the extracellular signal-related ki- nase is important for long-term increases in synaptic strength in the Aplysia nervous system. However, there is little known about the mechanism for the activation of the kinase in this system. We examined the activation of Aplysia extracellular signal-related kinase using a phosphopeptide antibody specific to the sites required for activation of the kinase. We found that phorbol esters led to a prolonged activation of extracellular signal-related kinase in sensory cells of the Aplysia nervous system. Surprisingly, inhibitors of protein kinase C did not block this activation. Serotonin, the physiological transmitter involved in long-term synaptic facilitation, also led to prolonged activation of extracellular signal-related kinase, but inhibitors of protein kinase A or protein kinase C did not block this activation. We examined whether the protein synthesis-dependent increase in excit- ability stimulated by phorbol esters was dependent on phor- bol ester activation of extracellular signal-related kinase, but increases in excitability were still seen in the presence of inhibitors of extracellular signal-related kinase activation. Our results suggest that prolonged phosphorylation of extra- cellular signal-related kinase in the Aplysia system is not mediated by either of the classic second messenger acti- vated kinases in this system, protein kinase A or protein kinase C and that extracellular signal-related kinase is not important for phorbol ester induced long-term effects on excitability. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: signal transduction, long-term facilitation, excitability, MAP kinases, learning and memory, diacylglycerol. In Aplysia, activation of gene expression is required for long-term facilitation of sensory to motorneuron synapses, but gene expression is not required for short-term facilita- tion of these synapses. A number of transcription factors have been implicated in the specific requirement for long- term facilitation (Alberini et al., 1994; Bartsch et al., 1995; Bartsch et al., 1998; Bartsch et al., 2000). The extracellular signal-related kinase (ERK) is an important signal that is thought to be important for the activation of the transcrip- tion factors that lead to the new gene expression required for long-term facilitation (Martin et al., 1997; Yamamoto et al., 1999). ERKs have also been implicated in long-term synaptic changes in many different systems other than Aplysia (Sweatt, 2001). ERK activity is important for the activation of the transcription factor cyclic AMP response element- binding protein (CREB) through activation of a CREB ki- nase in vertebrate neurons (Xing et al., 1996). Also, ERK can phosphorylate other transcription factors, and modu- lates synaptic strength through the phosphorylation of ion channels (Sweatt, 2001). There are a number of mechanisms by which ERKs can be activated downstream of neurotransmitter-gated G protein coupled receptors. In Aplysia, the neurotransmitter serotonin (5-HT) causes synaptic facilitation mainly through activation of the kinase protein kinase A (PKA) by cAMP and activation of the kinase protein kinase C (PKC) by diacylglycerol (DAG) (Byrne and Kandel, 1996). cAMP can activate ERK through either PKA phosphorylation of b-raf (Vossler et al., 1997) or directly through cAMP-gated guanine nucleotide exchange factors (GEFs) (de Rooij et al., 1998; Kawasaki et al., 1998). In both vertebrate and Aplysia neurons, cAMP and PKA activation are also im- portant in the translocation of ERK to the nucleus (Martin et al., 1997). PKC activation has been shown to be important for ERK activation through phosphorylation of the up- stream Raf kinase (Cai et al., 1997; Marais et al., 1998) and DAG can directly activate ERK through the DAG- activated GEF, RasGrp (Lorenzo et al., 2001). There are also other mechanisms by which ERK can be activated downstream of G protein-coupled receptor (Dikic and Blaukat, 1999; Pierce et al., 2001). We examined how 5-HT activates ERK in Aplysia sen- sory neurons. We find that sustained activation of ERK by 5-HT is independent of cAMP or PKC suggesting that *Corresponding author: Tel: +1-514-398-1486; fax: +1-514-398- 8106. E-mail address: wayne.sossin@mcgill.ca (W. Sossin). Abbreviations: CREB, cyclic AMP response element binding protein; DAG, diacylglycerol; ERK, extracellular signal-related kinase; 5-HT, 5-hydroxytryptamine or serotonin; GEF, guanine nucleotide exchange factor; PDBu, 4-phorbol ester 12,12 dibutyrate; PKA, protein kinase A; PKC, protein kinase C. Neuroscience 116 (2003) 13–17 0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. PII:S0306-4522(02)00566-3 13