Phactrs 1-4: A family of protein phosphatase 1 and actin regulatory proteins

Abstract

Protein phosphatase 1 (PP1) is a multifunctional enzyme with diverse roles in the nervous system, including regulation of synaptic activity and dendritic morphology. PP1 activity is controlled via association with a family of regulatory subunits that govern subcellular localization and substrate specificity. A previously undescribed class of PP1-binding proteins was detected by interaction cloning. Family members were also found to bind to cytoplasmic actin via Arg, Pro, Glu, and Leu repeat-containing sequences. The prototypical member of this family, phosphatase and actin regulator (phactr) 1 was a potent modulator of PP1 activity in vitro. Phactr-1 protein is selectively expressed in brain, where high levels were found in cortex, hippocampus, and striatum, with enrichment of the protein at synapses. Additional family members displayed highly distinct mRNA transcript expression patterns within rat brain. The current findings present a mechanism by which PP1 may be directed toward neuronal substrates associated with the actin cytoskeleton.

Fig. 1.

Identification of a PP1-binding protein. (A) Western blots prepared from 293T cell lysate fractions and immunoprecipitates demonstrating (Upper Left) expression of phactr yeast two-hybrid clone cDNA tagged with Flag epitope and (Lower Left) associated endogenous PP1. (A Right) Control fractions and immunoprecipitates prepared from cells transfected with empty expression vector (mock). (B) Modulation of purified phosphatase activity toward phosphorylase a after coincubation with increasing concentrations of either GST–phactr fusion protein or GST alone. Data are expressed as percentage activity in the absence of added recombinant protein.

Fig. 2.

Phactr interactions with PP1 and actin. (A) Abs raised against phactr detected a protein in rat brain lysate migrating with apparent molecular mass of ≈75 kDa. A protein of similar size was detected upon expression of phactr cDNA isolate in 293T cells. (B) Coimmunoprecipitation of phactr with PP1 and actin. Western blot analysis of protein levels in cell fractions (Left), phactr IP^+/- Ab-blocking peptide (Center), and PP1 IP^+/- Ab-blocking peptide (Right). Blot was reprobed sequentially with anti-phactr (Top), anti-actin (Middle), and anti-PP1 (Bottom) Abs. (C and D) Western blot analysis of coimmunoprecipitation reactions prepared by using cell lysates from 293T cells transfected with a series of phactr C-terminal truncation mutants (C) and a series of Ala-substitution mutants (D). IP was performed with anti-hemagglutinin Ab directed against N-terminal epitope tag. (E Upper) N-terminal mutants of phactr expressed in the yeast two-hybrid system. Interactions with PP1 and actin prey proteins were assessed by β-galactosidase activity in HF7c. (E Lower) Depiction of location of structural elements and protein binding sites within phactr. (F) Alignment of phactr RPEL repeats with consensus sequence for the motif (conserved domain smart00707.7). Conforming residues are shown in red. (G) Fluorescence image of N2a neuroblastoma cell transfected with full-length hemagglutinin epitope-tagged phactr. Red, rhodamine-phalloidin (F-actin); green, anti-hemagglutinin (phactr); blue, anti-PP1α. (Right Lower) Triple staining. (Scale bar, 10 μm.)

Fig. 3.

Expression pattern of phactr. (A) Northern blot analysis. DNA probe prepared from phactr cDNA (residues 541–1,061) was used to analyze rat poly(A)⁺ mRNA samples (Upper). Relative loading indicated by signal detected upon reprobing for glyceraldehyde-3 phosphate dehydrogenase (G3PDH) (Lower). (B) Western blot analysis of phactr protein levels detected in various tissues (Upper) and microdissected brain regions (Lower). Total protein, 50 μg per lane.

Fig. 4.

Immunocytochemical analysis of phactr-1 distribution in rat brain. (A) Coronal section revealing intense staining in striatum and olfactory tubercle, with moderate levels in septum and cortex. (Scale bar, 1 mm.) (B) Staining in CA1 was apparent in cell bodies and proximal dendrites but was absent from cell nuclei. (C) Staining in striatum also shows enrichment in cell bodies. (Scale bars, 25 μm.)

Fig. 5.

Phactr distribution within neurons. (A) Fluorescence image of a cultured hippocampal neuron transfected with N-terminally GFP-tagged phactr-1. (Scale bar, 20 μm.) (B) Western blot analysis for phactr-1 and NR1, indicating relative protein abundance in subcellular fractions. Protein (20 μg) loaded in the first six lanes: totals are 1,400 × g pellet (P1), 1,400 × g supernatant (S1), 13,800 × g pellet (P2), 13,800 × g supernatant (S2), and synaptic plasma membrane. The proteins (2 μg) loaded in the last two lanes were Triton X-100 extracted synaptic plasma membrane (PSD1) and Triton X-100/sarcosyl extracted PSD1 (PSD2).

Fig. 6.

Phactr family sequence homology and mRNA localization. (A) Amino acid similarity among members of the mouse phactr family. Alignment was performed with clustal iv algorithm. RPEL repeat sequences are underlined. (B) In situ hybridization of horizontal rat brain sections by using probes prepared for phactrs 1–4.