Phosphorylation of the Transactivation Domain of Pax6 by Extracellular Signal-regulated Kinase and p38 Mitogen-activated Protein Kinase

Abstract

The transcription factor Pax6 is required for normal development of the central nervous system, the eyes, nose, and pancreas. Here we show that the transactivation domain (TAD) of zebrafish Pax6 is phosphorylated in vitro by the mitogen-activated protein kinases (MAPKs) extracellular-signal regulated kinase (ERK) and p38 kinase but not by Jun N-terminal kinase (JNK). Three of four putative proline-dependent kinase phosphorylation sites are phosphorylated in vitro. Pax6 is a member of the paired box-containing Pax 1 gene family of transcription factors containing nine human (PAX1-PAX9) and murine (Pax1-Pax9) family members (1). The paired box was first discovered in Drosophila as encoding a conserved 125-128 amino acid paired domain unique to this family of developmental control genes (2). Pax6 was initially cloned from human (3), mouse (4), zebrafish (5), and quail (6). Subsequently, the Drosophila eyeless gene was shown to be a Pax6 homolog and Pax6 homologs have now been described in other invertebrates such as flatworm, ribbonworm, Caenorhabditis elegans, squid, sea urchin, and ascidian (reviewed in Ref. 7) as well as in amphioxus (8). Pax6 is expressed in the developing central nervous system, the eyes, nose, and pancreas in higher vertebrates (4, 5, 9, 10) and plays a pivotal role in the development of these organs (7,11,12). Loss of a functional Pax6 allele results in the Mendelian syndromes aniridia, Peter's anomaly, and congenital cataracts in man (13) and Small eye in rodents (14). Pax6 acts high up in the regulatory hierarchy controlling eye development in both vertebrates and invertebrates (reviewed in Ref. 15). Eyeless, ribbonworm-, squid-, ascidian-, zebrafish-, or mouse Pax6 are all able to induce ectopic eyes in Drosophila upon targeting their expression to different imaginal discs (16 -20). We have recently found that zebrafish contain two Pax6 genes, Pax6.1 and Pax6.2, which are expressed in both overlapping and distinct regions during development of the eyes and the central nervous system. Both these genes are able to induce ectopic eyes in Drosophila (18).The paired domain is a bipartite DNA-binding domain containing an N-and a C-terminal subdomain each with a helixturn-helix motif (21, 22). Pax6, like Pax3, Pax4, and Pax7, also harbors a second DNA-binding domain, the paired-type homeodomain (2, 7, 23). In Pax6 this domain is separated from the N-terminal located paired domain by a flexible, acidic linker region (5, 7). The region C-terminal to the homeodomain is enriched in proline, serine, and threonine residues (PST-rich) and acts as a transactivation domain (TAD) (24 -27).It has previously been shown that quail Pax6 proteins expressed in the neuroretina are phosphoproteins and phosphoamino acid analysis revealed phosphoserine and a minor proportion of phosphothreonine (28). The activity of many transcription factors is regulated in a rapid and reversible manner by specific phosphorylation events mediated by protein kinases acting in signaling cascades initiated by extracellular stimuli (r...

B Cell Receptor-ERK1/2 Signal Cancels PAX5-Dependent Repression of BLIMP1 through PAX5 Phosphorylation: A Mechanism of Antigen-Triggering Plasma Cell Differentiation

“…p38 phosphorylate PAX6 at the same sites: serines 376 and 413 and threonine 323 (30). This phosphorylation enhances the transcriptional activities of PAX family proteins.…”

Sumoylation activates the transcriptional activity of Pax-6, an important transcription factor for eye and brain development

“…S1A). This domain mediates activation of Pax-6 through phosphorylation by p38 MAP kinase and homeodomaininteracting protein kinase 2 (15,16). Several phosphorylation sites have been identified in the PST domain of human and zebrafish Pax-6.…”

Protein Phosphatase-1 Modulates the Function of Pax-6, a Transcription Factor Controlling Brain and Eye Development

“…These features suggest that Pax-6, as a transcription factor, could be modulated by post-translational modifications, such as phosphorylation and dephosphorylation. Indeed, it has been shown that the function of Pax-6 can be modulated by several kinases, including p38, ERK (34), and homeodomain-interacting protein kinase 2 (35). In the present study, we have demonstrated that although both PP-1 and PP-2A are able to dephosphorylate Pax-6 in the in vitro dephosphorylation assays, in the immunoprecipitation-linked Western blot analysis, PP-2A and Pax-6 failed to form interactive complex, and thus, it is unlikely that PP-2A dephosphorylates Pax-6 in vivo.…”

“…1B). It has been shown that phosphorylation of these sites in Pax-6 is carried out by p38, ERK (34), and homeodomain-interacting protein kinase 2 (35). On the other hand, dephosphorylation of Pax-6 remains largely unknown.…”