ORIGINAL ARTICLE ERK MAP kinase signaling in post-mortem brain of suicide subjects: differential regulation of upstream Raf kinases Raf-1 and B-Raf Y Dwivedi 1 , HS Rizavi 1 , RR Conley 2 and GN Pandey 1 1 Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA and 2 Maryland Psychiatric Research Center, Baltimore, MD, USA The Raf kinases Raf-1 and B-Raf are upstream activators of the extracellular signal-regulated kinase (ERK)-signaling pathway and therefore participates in many physiological functions in brain, including neuronal survival and synaptic plasticity. Previously, we observed that activation of ERK-1/2, the downstream component of ERK signaling, is significantly reduced in post-mortem brain of suicide victims. The present study was undertaken to further examine whether suicide brain is also associated with abnormalities in upstream molecules in ERK signaling. The study was performed in prefrontal cortex (PFC) and hippocampus obtained from 28 suicide victims and 21 normal controls. mRNA levels of Raf-1, B-Raf, and cyclophilin were measured by quantitative RT-PCR. Protein levels of Raf-1 and B-Raf were determined by Western blot, whereas their catalytic activities were determined by immunoprecipitation and enzymatic assays. It was observed that the catalytic activity of B-Raf was significantly reduced in PFC and hippocampus of suicide subjects. This decrease was associated with a decrease in its protein, but not mRNA, level. On the other hand, catalytic activity, and mRNA and protein levels, of Raf-1 were not altered in post-mortem brain of suicide subjects. The observed changes were not related to confounding variables; however, Raf-1 showed a negative correlation with age. Also, the changes in B-Raf were present in all suicide subjects, irrespective of psychiatric diagnosis. Our results of selective reduction in catalytic activity and expression of B-Raf but not Raf-1 suggest that B-Raf may be playing an important role in altered ERK signaling in brain of suicide subjects, and thus in the pathophysiology of suicide. Molecular Psychiatry (2006) 11, 86–98. doi:10.1038/sj.mp.4001744; published online 20 September 2005 Keywords: human; prefrontal cortex; hippocampus; signal transduction; competitive RT-PCR; immunolabeling; catalytic activity; depression Introduction Mitogen-activated protein/extracellular signal-regu- lated kinase (ERK) is a family of serine/threonine protein kinases that are critical for several neuronal functions regulated by growth factors/neurotrophins, and their receptors associated with tyrosine kinases. These include neuronal differentiation and survival of neurons during development, as well as survival and adaptive responses of mature neurons including synaptic plasticity and learning and memory. 1–6 The prerequisite for ERK activation involves the phos- phorylation of ERKs in a cascade of events that leads to their subsequent translocation from the cytoplasm to the nucleus, where they regulate gene expression by phosphorylating specific transcription factors, such as Elk-1 and cAMP-response element binding protein. 7 The activation of ERKs involves a cascade of events in which a small GTP-binding protein, Ras, recruits Raf kinases to the plasma membrane. 8 Raf kinases then phosphorylate and activate ERK kinase, or MEK, which in turn phosphorylates and activates ERKs. 9 The sequential nature of this cascade thus provides multiple points at which the responses can be regulated by phosphorylation, which allows tremendous amplification of extracellular signals. A recent hypothesis proposes that depression and stress, two important factors that contribute to suicidal behavior, are associated with impairments in neural/structural plasticity, neurogenesis, and cellular resilience. 10–13 Structural abnormalities in brain of affective disorder patients, during stress, and in suicide subjects have been reported. 14–20 Since synaptogenesis and synaptic remodeling are influ- enced by neurotrophic factors and their mediated ERK signaling through tyrosine receptor kinases, 21 a number of studies have examined the role of neurotrophins in depression, 22 during stress 23,24 and in the pathophysiology of suicide; 25 and that BDNF, one of the neurotrophins, may ameliorate depression. 26 Received 25 May 2005; revised 22 July 2005; accepted 9 August 2005; published online 20 September 2005 Correspondence: Dr Y Dwivedi, Department of Psychiatry, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA. E-mail: ydwivedi@psych.uic.edu Molecular Psychiatry (2006) 11, 86–98 & 2006 Nature Publishing Group All rights reserved 1359-4184/06 $30.00 www.nature.com/mp