Identification and characterization of AtI-2, anArabidopsishomologue of an ancient protein phosphatase 1 (PP1) regulatory subunit

Abstract

PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins.

Activation of Mitochondrial Protein Phosphatase SLP2 by MIA40 Regulates Seed Germination

“…Fluorescent constructs were subsequently created using the binary vectors pB7RWG2 (cRFP) and pK7RWG2 (cGFP; https://gateway.psb.ugent.be/), while TAP clones were created using pYL436 (Rubio et al, 2005). Transformation of each construct into Agrobacterium tumefaciens strain GV3101 (A. tumefaciens) was performed to facilitate stable integration of each construct into either Arabidopsis plants (Zhang et al, 2006) or cell culture (Templeton et al, 2011).…”

“…Positive transformants were screened via immunoblotting with anti-Myc IgG (ICL). Arabidopsis cell culture was subsequently transitioned from light to dark over a 2 week period, with subculturing performed every 7 d. TAP pull-downs from dark cell culture were performed as previously described (Templeton et al, 2011) using an extract from 50 g of cells per pull-down, while pull-downs from roots were performed using 5 g of tissue. Subsequent washing of tagged protein bound Ni-NTA with 10 mL of triethylammonium bicarbonate (pH 8.5) was performed prior to on-bead, overnight (18 h) proteolysis using 750 ng of trypsin (Promega) at 30°C.…”

“…Sample digestion, processing, mass spectrometry, and data analysis were performed as in Templeton et al (2011). In addition to GFP-TAP pull-downs controlling for experimentation involving cell culture, AtSLP1 offered a control for AtSLP2 pull-downs from root tissue, as AtSLP1 is not expressed in nongreen tissues and is the closest ortholog of AtSLP2 (Uhrig and Moorhead, 2011).…”

Okadaic acid and microcystin insensitive PPP-family phosphatases may represent novel biotechnology targets

“…Conversely, SLP phosphatase containing bacteria and fungi were found to possess microcystin, 9,10 okadaic acid 11 and inhibitor-2 protein. 12 Inhibitor-2 protein is a highly conserved, genomically encoded protein interactor specific to the type one (PP1) protein phosphatases. 12 As an innately flexible protein, inhibitor-2 exerts its inhibitory effect through a combination of induced conformational changes and direct occlusion of the PP1 active site.…”

“…12 Inhibitor-2 protein is a highly conserved, genomically encoded protein interactor specific to the type one (PP1) protein phosphatases. 12 As an innately flexible protein, inhibitor-2 exerts its inhibitory effect through a combination of induced conformational changes and direct occlusion of the PP1 active site. 13,14 Microcystin and okadaic acid however, are two representatives of a chemically diverse and naturally occurring group of small molecule toxins that target the PPP-family enzymes.…”