Abstract
The RdgC/PPEF family of serine/threonine protein phosphatases is distinguished by the presence of C-terminal EF-hands and neuron-specific expression, including frequent expression in primary sensory neurons. Here we report that the sole Caenorhabditis elegans PPEF (CePPEF) homolog is also highly expressed in primary sensory neurons and is not found outside the nervous system. Neurons expressing CePPEF include the ciliary chemosensory neurons AWB and AWC; and within these neurons, CePPEF is highly enriched in the sensory cilia. In transgenic C. elegans and in transfected 293 cells, CePPEF is membrane-associated, and the N terminus of CePPEF is necessary and sufficient for this membrane association. [(3)H]Myristate and [(3)H]palmitate labeling studies in 293 cells demonstrated that this association was mediated by myristoylation at Gly(2) and palmitoylation at Cys(3). Introducing the G2A or C3S mutation into CePPEF greatly reduced membrane association in 293 cells and in transgenic nematodes. A recombinant C-terminal fragment of CePPEF containing two putative EF-hands bound between one and two Ca(2+) ions/protein, and mutation of residues presumed to ligand calcium in the two putative EF-hands led to diminished calcium binding. These results establish the first direct evidence for fatty acylation and calcium binding of a PPEF family member and demonstrate a remarkable conservation of sensory neuron expression among the members of this distinctive family of protein phosphatases.
Highlights
Phosphorylation and dephosphorylation of serine and threonine residues are known to be important in several types of sensory neurons
To assess the subcellular localization of the Caenorhabditis elegans PPEF (CePPEF) protein and to determine whether additional transcriptional regulatory elements exist in intronic sequences, a transgene was constructed in which a 9.5-kb genomic DNA fragment extending from 3 kb 5Ј of the start codon to a point 32 base pairs upstream of the CePPEF stop codon in exon 7 was fused in-frame to GFP (FL-GFP) (Fig. 1)
This work represents the first characterization of CePPEF, the sole member of the PPEF family of serine/threonine protein phosphatases encoded within the C. elegans genome
Summary
CDNA and Genomic Sequences— phage clones encompassing the F23H11.8 (CePPEF) locus were isolated from a C. elegans genomic DNA library and used to prepare transgene constructs. [3H]Palmitate was added to Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 with 5% calf serum at a specific activity of 1000 Ci/ml [27], and cells were incubated in labeling medium for 1 h. Calcium binding at each Ca2ϩ concentration was assayed in triplicate; a control reaction without protein was analyzed for each Ca2ϩ concentration and subtracted as background. Binding reactions were added to the pre-equilibrated Sephadex G-50 spin column and centrifuged at 731 ϫ g for 1 min at 22 °C. Calcium binding at each Ca2ϩ concentration was assayed in nine independent Sephadex G-50 spin columns; three control reactions without protein were analyzed for each Ca2ϩ concentration and subtracted as background. Adjustment of the numerator by a factor of 0.675 is designed to set maximal binding to the binding observed at 1 mM Ca2ϩ and accounts for the lowered maximal binding due to an estimated 80 –90% purity of the fusion protein sample and retention of 10 –20% of the fusion protein on the Sephadex G-50 spin column
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