Research Article A role for prolyl isomerase PIN1 in the phosphorylation-dependent modulation of PRRXL1 function Ricardo Soares-dos-Reis 1,2,3,4,5, *, Ana Sofia Pessoa 1,3,6, *, Ana Filipa Dias 1,2,3 , Miguel Falcão 1,2,3 , Mariana Raimundo Matos 1,2,3 , Rui Vitorino 7,8 , Filipe Almeida Monteiro 1,2,3 , Deolinda Lima 1,2,3 and Carlos Reguenga 1,2,3 1 Departamento de Biomedicina, Unidade de Biologia Experimental, Faculdade de Medicina da Universidade do Porto, Porto, Portugal; 2 i3S — Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; 3 IBMC — Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; 4 Departamento de Neurociências Clínicas e Saúde Mental, Faculdade de Medicina da Universidade do Porto, Porto, Portugal; 5 Serviço de Neurologia, Centro Hospitalar de São João, Porto, Portugal; 6 Serviço de Medicina, Centro Hospitalar do Médio Ave, Vila Nova de Famalicão, Portugal; 7 Departamento de Cirurgia e Fisiologia, Faculdade de Medicina da Universidade do Porto, Porto, Portugal; and 8 Departamento de Ciências Médicas, iBiMED — Instituto de Biomedicina da Universidade de Aveiro, Aveiro, Portugal Prrxl1 encodes for a paired-like homeodomain transcription factor essential for the correct establishment of the dorsal root ganglion — spinal cord nociceptive circuitry during development. Prrxl1-null mice display gross anatomical disruption of this circuitry, which translates to a markedly diminished sensitivity to noxious stimuli. Here, by the use of an immunoprecipitation and mass spectrometry approach, we identify five highly con- served phosphorylation sites (T110, S119, S231, S233 and S251) in PRRXL1 primary structure. Four are phospho-S/T-P sites, which suggest a role for the prolyl isomerase PIN1 in regulating PRRXL1. Accordingly, PRRXL1 physically interacts with PIN1 and dis- plays diminished transcriptional activity in a Pin1-null cell line. Additionally, these S/T-P sites seem to be important for PRRXL1 conformation, and their point mutation to alanine or aspartate down-regulates PRRXL1 transcriptional activity. Altogether, our findings provide evidence for a putative novel role of PIN1 in the development of the nociceptive system and indicate phosphorylation-mediated conformational changes as a mechanism for regulating the PRRXL1 role in the process. Introduction Neuronal heterogeneity arises from the action of master selector genes, often switching between mutu- ally exclusive terminal fates. This terminal differentiation program includes receptors, cell-adhesion molecules and neurotransmitter machinery, which provide neurons with their unique subtype-specific properties. The combinatorial action of transcription factors is the basis for the establishment of neuronal diversity [1]. However, it is still largely unknown how transcription factors interact and regulate downstream genes as to set the molecular signature of a particular neuronal population. Tlx3 is necessary for early specification (E10.5) of an excitatory over inhibitory interneuron fate, and later for the differentiation of the glutamatergic neuronal identity in the dorsal spinal cord (dSC) [2]. Prrxl1, on its turn, appears to be important to maintain and initiate the terminal differentiation of a major portion of the glutamatergic nociceptive spinal neuronal population and the establishment of the respective dorsal root ganglion (DRG) connections [3,4]. Prrxl1-null mouse embryos exhibit impaired morphogenesis of the spinal cord dorsal horn, with ablation of most superficial dorsal horn glutamatergic neurons, and spatiotemporal defects in dorsal horn nociceptor projection [3]. These animals also present marked reduction in postnatal sensitivity to noxious stimuli, with mostly unaltered proprioception and sensorimotor functions [4]. Prrxl1 expression is residual in adult life, but an increase is observed at the DRG during inflammatory pain, suggesting a role for Prrxl1 in noci- ceptor sensitization [5]. *These authors contributed equally to this work. Accepted Manuscript online: 3 January 2017 Version of Record published: 20 February 2017 Received: 13 June 2016 Revised: 10 December 2016 Accepted: 3 January 2017 © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society 683 Biochemical Journal (2017) 474 683–697 DOI: 10.1042/BCJ20160560 Downloaded from https://portlandpress.com/biochemj/article-pdf/474/5/683/690523/bcj-2016-0560.pdf by guest on 30 May 2020