Identification and Characterization of a Novel Human PP1 Phosphatase Complex

Abstract

Mammalian Wdr82 is a regulatory component of the Setd1a and Setd1b histone H3-lysine 4 methyltransferase complexes and is implicated in the tethering of Setd1 complexes to transcriptional start sites of active genes. In the studies reported here, immunoprecipitation and mass spectrometry analyses reveal that Wdr82 additionally associates with multiple protein complexes, including an RNA polymerase II complex, four distinct histone H3-Lys 4 methyltransferase complexes, protein phosphatase 1 (PP1)-associated proteins, a chaperonin-containing Tcp1 complex, and other uncharacterized proteins. Further characterization of the PP1-associated proteins identified a stable multimeric complex composed of regulatory subunits PNUTS, Tox4, and Wdr82 and a PP1 catalytic subunit (denoted as the PTW/PP1 phosphatase complex). The PTW/ PP1 complex exhibits in vitro phosphatase activity in a PP1-dependent manner. Analysis of protein-protein interactions reveals that PNUTS mediates phosphatase complex formation by providing a binding platform to each component. The PNUTS and Tox4 subunits are predominantly associated with the PTW/PP1 phosphatase complex in HEK293 cells, and the integrity of this complex remains intact throughout cell cycle progression. Inducible expression of a PP1 interaction-defective form of PNUTS (W401A) or small interfering RNA-mediated depletion of PNUTS in HEK293 cells causes cell cycle arrest at mitotic exit and apoptotic cell death. PNUTS (W401A) shows normal association with chromosomes but causes defects in the process of chromosome decondensation at late telophase. These data reveal that mammalian Wdr82 functions in a variety of cellular processes and reveal a potential role of the PTW/PP1 phosphatase complex in the regulation of chromatin structure during the transition from mitosis into interphase. Protein phosphatase 1 (PP1)2 is a serine/threonine protein phosphatase involved in diverse cellular processes, such as transcription, replication, pre-mRNA splicing, protein synthesis, muscle contraction, carbohydrate metabolism, neuronal signaling, cell survival, and cell cycle progression (1-3). Mammals express three PP1 catalytic isoforms, PP1␣, PP1␥, and PP1␤/, which show distinct subcellular localization patterns (4, 5). PP1 catalytic isoforms do not exist freely in cells but rather associate with regulatory subunits to form distinct multimeric holoenzymes. In general, PP1 regulatory subunits function as signaling modules by regulating the enzymatic activity or targeting of catalytic subunits to specific substrates (6, 7). PP1 is involved in cell cycle progression, especially during mitosis and mitotic exit, and dysregulation of PP1 activity causes mitotic arrest or deficient cytokinesis in mammals (3,8,9). PP1 is associated with multiple mitotic structures, such as chromosomes, centrosomes, and spindles (4, 8, 10), and is implicated as a major phosphatase acting on phosphoproteins at mitotic exit (11-14). However, the specific regulatory or targeting subunits required for this activity are not well und...

Copy number variants in patients with short stature

“…Out of the six genes outside the overlapping region with patient #258497 CHD8 and TOX4 appear potential candidate genes. 41,42 Case II.22/mi.5 has a 22q deletion containing only the distal part of the common 22q11 deletion syndrome (Velocardiofacial/DiGeorge syndrome). His mother does not carry the duplication, and DNA from the father is not available.…”

A quantitative telomeric chromatin isolation protocol identifies different telomeric states

“…In addition, we identified peptides from several other reported telomereassociated components and factors known to assemble with telomere-associated components (Supplementary Table S1). This includes the telomere end processing enzymes Apollo, Mre11, Nbs1 and Rad50; ORC complex components, DNA polymerase d, the WRN interacting protein WRNIP1, which is required for efficient telomere replication 13 ; PNUTS 16 and subunits of the PNUTS containing PTW/PP1 phosphatase complex, which had not been previously characterized at telomeres 17 .…”

Biophysical Analysis of the N-Terminal Domain from the Human Protein Phosphatase 1 Nuclear Targeting Subunit PNUTS Suggests an Extended Transcription Factor TFIIS-Like Fold

“…It forms a ternary complex with PP1, Tox4 and WDR82 that targets PP1 to the nucleus [3][4][5][6][7][8][9][10], and further interacts with the tumour suppressor phosphatase and tensin homolog PTEN [11].…”

“…At the molecular level, PNUTS is a largely unstructured protein that contains two small folded domains, located at each of its termini. The N-terminal domain is predicted to be similar to the N-terminal transcription factor IIS (TFIIS) LW domain (so-called by the presence of invariant leucine and tryptophan residues; [12]) and binds to Tox-4 [6] and PTEN [11]. Such TFIIS LW domains are small four-helix bundles that are present in transcription factors such as MED26 and elongin A [12].…”

“…Such TFIIS LW domains are small four-helix bundles that are present in transcription factors such as MED26 and elongin A [12]. They are part of the larger TFIIS module that engages RNApolymerase II [13,14] and Tox-4 [6]. The PNUTS C-terminal region contains a zinc finger domain implying a possible interaction with nucleic acids [14], although this domain in PNUTS is not known to bind either RNA or DNA.…”