Structural and Biochemical Characterization of the Two Drosophila Low Molecular Weight-Protein Tyrosine Phosphatases DARP and Primo-1.

The Drosophila genome contains four low molecular weightprotein tyrosine phosphatase (LMW-PTP) members: Primo-1, Primo-2, CG14297, and CG31469. The lack of intensive biochemical analysis has limited our understanding of these proteins. Primo-1 and CG31469 were previously classified as pseudophosphatases, but CG31469 was also suggested to be a putative protein arginine phosphatase. Herein, we present the crystal structures of CG31469 and Primo-1, which are the first Drosophila LMW-PTP structures. Structural analysis showed that the two proteins adopt the typical LMW-PTP fold and have a canonically arranged P-loop. Intriguingly, while Primo-1 is presumed to be a canonical LMW-PTP, CG31469 is unique as it contains a threonine residue at the fifth position of the P-loop motif instead of highly conserved isoleucine and a characteristically narrow active site pocket, which should facilitate the accommodation of phosphoarginine. Subsequent biochemical analysis revealed that Primo-1 and CG31469 are enzymatically active on phosphotyrosine and phosphoarginine, respectively, refuting their classification as pseudophosphatases. Collectively, we provide structural and biochemical data on two Drosophila proteins: Primo-1, the canonical LMW-PTP protein, and CG31469, the first investigated eukaryotic protein arginine phosphatase. We named CG31469 as DARP, which stands for Drosophila ARginine Phosphatase.

The transcription factor PITX1 drives astrocyte differentiation by regulating the SOX9 gene.

Astrocytes perform multiple essential functions in the developing and mature brain, including regulation of synapse formation, control of neurotransmitter release and uptake, and maintenance of extracellular ion balance. As a result, astrocytes have been implicated in the progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite these critical functions, the study of human astrocytes can be difficult because standard differentiation protocols are time-consuming and technically challenging, but a differentiation protocol recently developed in our laboratory enables the efficient derivation of astrocytes from human embryonic stem cells. We used this protocol along with microarrays, luciferase assays, electrophoretic mobility shift assays, and ChIP assays to explore the genes involved in astrocyte differentiation. We demonstrate that paired-like homeodomain transcription factor 1 (PITX1) is critical for astrocyte differentiation. PITX1 overexpression induced early differentiation of astrocytes, and its knockdown blocked astrocyte differentiation. PITX1 overexpression also increased and PITX1 knockdown decreased expression of sex-determining region Y box 9 (SOX9), known initiator of gliogenesis, during early astrocyte differentiation. Moreover, we determined that PITX1 activates the SOX9 promoter through a unique binding motif. Taken together, these findings indicate that PITX1 drives astrocyte differentiation by sustaining activation of the SOX9 promoter.