Structural Analysis of the Protein Phosphatase 1 Docking Motif: Molecular Description of Binding Specificities Identifies Interacting Proteins

During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein phosphatase 1 (PP1). However, the regulatory mechanisms underlying the aforementioned hierarchical signaling cascade during mitotic chromosome segregation have remained elusive. Sds22 is a conserved regulator of PP1 activity, but how it regulates PP1 activity in space and time during mitosis remains elusive. Here we show that Sds22 is a novel and cognate substrate of PLK1 in mitosis, and the phosphorylation of Sds22 by PLK1 elicited an inhibition of PP1-mediated dephosphorylation of Aurora B at threonine 232 (Thr 232 ) in a dose-dependent manner. Overexpression of a phosphomimetic mutant of Sds22 causes a dramatic increase in mitotic delay, whereas overexpression of a non-phosphorylatable mutant of Sds22 results in mitotic arrest. Mechanistically, the phosphorylation of Sds22 by PLK1 strengthens the binding of Sds22 to PP1 and inhibits the dephosphorylation of Thr 232 of Aurora B to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling hierarchy that orchestrates dynamic protein phosphorylation and dephosphorylation of critical mitotic regulators during chromosome segregation to guard chromosome stability.

Section: Discussionmentioning

confidence: 99%

Phosphorylation of PP1 Regulator Sds22 by PLK1 Ensures Accurate Chromosome Segregation

Duan

Wang²,

Wang

et al. 2016

“…3b. domain, the sequence that docks at the RVXF site is KSQKW. Thus, the sequence degeneracy at the RVXF site is greater than previously anticipated (44,47), and in I-2 an Ala at the Val position can be tolerated (Fig. 3).…”

Section: Structure Of the Pp1⅐inhibitor-2 Complexmentioning

confidence: 93%

Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2

Hurley

Yang

Zhang

et al. 2007

180

246

The functional specificity of type 1 protein phosphatases (PP1) depends on the associated regulatory/targeting and inhibitory subunits. To gain insights into the mechanism of PP1 regulation by inhibitor-2, an ancient and intrinsically disordered regulator, we solved the crystal structure of the complex to 2.5Å resolution. Our studies show that, when complexed with PP1c, I-2 acquires three regions of order: site 1, residues 12-17, binds adjacent to a region recognized by many PP1 regulators; site 2, amino acids 44 -56, interacts along the RVXF binding groove through an unsuspected sequence, KSQKW; and site 3, residues 130 -169, forms ␣-helical regions that lie across the substratebinding cleft. Specifically, residues 148 -151 interact at the catalytic center, displacing essential metal ions, accounting for both rapid inhibition and slower inactivation of PP1c. Thus, our structure provides novel insights into the mechanism of PP1 inhibition and subsequent reactivation, has broad implications for the physiological regulation of PP1, and highlights common inhibitory interactions among phosphoprotein phosphatase family members.

“…These subunits bind to a degenerate RVXF motif present in their interacting proteins. Binding to these regulatory subunits is regulated by phosphorylation (54,60). Several proteins, including inhibitor-1, inhibitor-2, DARPP-32, and NIPP1 (nuclear inhibitor of PP1), that block PP1 activity through tight binding have been identified (54,61).…”

Section: Phosphatases Acting On Splicing Regulatory Proteinsmentioning

confidence: 99%

Regulation of Alternative Splicing by Reversible Protein Phosphorylation

Stamm

2008

226

188

The vast majority of human protein-coding genes are subject to alternative splicing, which allows the generation of more than one protein isoform from a single gene. Cells can change alternative splicing patterns in response to a signal, which creates protein variants with different biological properties. The selection of alternative splice sites is governed by the dynamic formation of protein complexes on the processed pre-mRNA. A unique set of these splicing regulatory proteins assembles on different pre-mRNAs, generating a "splicing" or "messenger ribonucleoprotein code" that determines exon recognition. By influencing protein/protein and protein/RNA interactions, reversible protein phosphorylation modulates the assembly of regulatory proteins on pre-mRNA and therefore contributes to the splicing code. Studies of the serine/arginine-rich protein class of regulators identified different kinases and protein phosphatase 1 as the molecules that control reversible phosphorylation, which controls not only splice site selection, but also the localization of serine/arginine-rich proteins and mRNA export. The involvement of protein phosphatase 1 explains why second messengers like cAMP and ceramide that control the activity of this phosphatase influence alternative splicing. The emerging mechanistic links between splicing regulatory proteins and known signal transduction pathways now allow in detail the understanding how cellular signals modulate gene expression by influencing alternative splicing. This knowledge can be applied to human diseases that are caused by the selection of wrong splice sites. Regulation of Splice Site SelectionMost protein-coding genes contain introns that are removed in the nucleus by RNA splicing during pre-mRNA processing. Parts of the pre-mRNA can be either included or excluded in the mature mRNA, which is achieved by alternative splicing.This process is much more widely used than previously thought and was recently estimated to affect between 74 and 88% of human genes (1, 2), but the exact number still needs to be determined. Because most alternative exons encode protein modules, their alternate use allows multiple proteins to be generated from a single gene, which increases the coding potential of the genome. Alternative splicing generates protein isoforms with different biological properties, such as a change in protein/ protein interaction, subcellular localization, or catalytic ability (3). More than one quarter of alternative exons introduce premature stop codons in their mRNAs. This can result either in the formation of truncated proteins or in the degradation of the mRNA by nonsense-mediated decay. Recent array analyses indicate that, although frequently found, alternative exons with premature stop codons appear to be generally present only in low abundance, which questions their role as a general shutoff mechanism for protein production (4, 5).Splice site recognition and pre-mRNA splicing are dynamic processes that involve constant remodeling of proteins and ribonuclear protein pa...