Ser119 phosphorylation modulates the activity and conformation of PRRXL1, a homeodomain transcription factor

Abstract

PRRXL1 [paired related homeobox-like 1; also known as DRG11 (dorsal root ganglia 11)] is a paired-like homeodomain transcription factor expressed in DRG and dSC (dorsal spinal cord) nociceptive neurons. PRRXL1 is crucial for the establishment and maintenance of nociceptive circuitry, as Prrxl1(-/-) mice present neuronal loss, reduced pain sensitivity and failure to thrive. In the present study, we show that PRRXL1 is highly phosphorylated in vivo, and that its multiple band pattern on electrophoretic analysis is the result of different phosphorylation states. PRRXL1 phosphorylation appears to be differentially regulated along the dSC and DRG development and it is mapped to two functional domains. One region comprises amino acids 107-143, whereas the other one encompasses amino acids 227-263 and displays repressor activity. Using an immunoprecipitation-MS approach, two phosphorylation sites were identified, Ser¹¹⁹ and Ser²³⁸. Phosphorylation at Ser¹¹⁹ is shown to be determinant for PRRXL1 conformation and transcriptional activity. Ser¹¹⁹ phosphorylation is thus proposed as a mechanism for regulating PRRXL1 function and conformation during nociceptive system development.

Paired related homeobox protein‐like 1 (Prrxl1) controls its own expression by a transcriptional autorepression mechanism

“…2B), with Prrxl1 displaying the strongest effect. Accordingly, recent data showed that Prrxl1 has a repressor domain spanning amino acids 227-263 of Prrxl1, which is not present in Prrxl1b [28]. No repression was detected when using a region spanning Prrxl1 alternative promoters downstream of the TATA box [À604/À50].…”

Dual role of Tlx3 as modulator of Prrxl1 transcription and phosphorylation

“…Recently, we have shown that Prrxl1 is differentially phosphorylated in the dorsal spinal cord and DRG along development [19]. The increase in phosphorylation state is accompanied by conformational changes resulting in a multiple band pattern on electrophoretic analysis [19].…”

“…To better understand the regulatory mechanisms implicated in Tlx3 and Brn3a control of Prrxl1 expression, we performed gain-and loss-offunction experiments in ND7/23 cells, a DRG-derived cell line previously shown to be a suitable model for studying Prrxl1 [9,19]. Given that promoters P1 and P2 share many features, we decided to analyse these DNA regions as a single module.…”

“…Expression vectors previously constructed were pRSK-Brn3a (a gift from Dr. Mengqing Xiang), pcDNA3.3-Tlx3 [9], pcDNA3.3-Prrxl1 [19], pcDNA3.3-HA-Prrxl1 [19] and pCMVβ (Clontech). The sequences used to clone Tlx3-FLAG or the truncated variants (Tlx3 38-291 -FLAG, Tlx3 76-291 -FLAG, Tlx3 111-291 -FLAG, Tlx3 156-291 -FLAG, Tlx3 1-256 -FLAG and Tlx3 1-224 -FLAG) into pCDNA3.3 were PCR amplified from pcDNA3.3-Tlx3 with primers depicted in Table 1.…”

“…Isoelectric focusing was performed on a Protean i12 Cell (Bio-Rad) using 11 cm immobilized pH gradient strips pH 3-10 nonlinear (Bio-Rad) for 38,000 Vh and held at 750 V as previously described [19]. Afterwards, SDS-polyacrylamide gels (12%) were run, and Western blotting was performed with home-made rabbit anti-Prrxl1 antibody [10].…”