Greg B. G. Moorhead

14-3-3 Proteins: A Number of Functions for a Numbered Protein

Many signal transduction events are orchestrated by specific interactions of proteins mediated through discrete phosphopeptide-binding motifs. Although several phosphospecific-binding domains are now known, 14-3-3s were the first proteins recognized to specifically bind a discrete phosphoserine or phosphothreonine motif. The 14-3-3 proteins are a family of ubiquitously expressed, exclusively eukaryotic proteins with an astonishingly large number of binding partners. Consequently, 14-3-3s modulate an enormous and diverse group of cellular processes. The effects of 14-3-3 proteins on their targets can be broadly defined using three categories: (i) conformational change; (ii) physical occlusion of sequence-specific or structural protein features; and (iii) scaffolding. This review will describe the current state of knowledge on 14-3-3 proteins, highlighting several important advances, and will attempt to provide a framework by which 14-3-3 functions can be understood. Signal transduction events can be regulated both by posttranslational modifications and by protein-protein interactions. 14-3-3 proteins are critically involved in both of these processes. The 14-3-3s, originally catalogued as small, abundant brain proteins, are expressed as multiple isoforms in all eukaryotic cells and are now known to recognize and to bind to distinct phosphoserine or phosphothreonine motifs on target proteins. Their binding partners include key proteins involved in metabolism, cell cycle control, the DNA damage response, transcription, protein synthesis, and apoptosis. This STKE Review with 2 figures and 127 references describes 14-3-3 proteins and highlights how these simple proteins have profound effects on the regulation of a vast number of cellular events.

Emerging roles of nuclear protein phosphatases.

The phosphorylation state of any protein represents a balance of the actions of specific protein kinases and protein phosphatases. Many protein phosphatases are highly enriched in, or exclusive to, the nuclear compartment, where they dephosphorylate key substrates to regulate various nuclear processes. In this review we will discuss recent findings that define the role of nuclear protein phosphatases in controlling transforming growth factor-β (TGFβ) and bone-morphogenetic protein (BMP) signalling, the DNA-damage response, RNA processing, cell-cycle progression and gene transcription.

Autophosphorylation of ataxia‐telangiectasia mutated is regulated by protein phosphatase 2A

Ionizing radiation induces autophosphorylation of the ataxia‐telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A‐like activity in vitro. OA did not induce γ‐H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double‐strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A‐like protein phosphatase activity. Moreover, we show that IR induces phosphorylation‐dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.

Repo-Man recruits PP1γ to chromatin and is essential for cell viability

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase regulating many cellular processes. PP1α and -γ are closely related isoforms with distinct localization patterns, shown here by time-lapse microscopy of stably expressed fluorescent protein fusions. A pool of PP1γ is selectively loaded onto chromatin at anaphase. Using stable isotope labeling and proteomics, we identified a novel PP1 binding protein, Repo-Man, which selectively recruits PP1γ onto mitotic chromatin at anaphase and into the following interphase. This approach revealed both novel and known PP1 binding proteins, quantitating their relative distribution between PP1α and -γ in vivo. When overexpressed, Repo-Man can also recruit PP1α to chromatin. Mutating Repo-Mans PP1 binding domain does not disrupt chromatin binding but abolishes recruitment of PP1 onto chromatin. RNA interference–induced knockdown of Repo-Man caused large-scale cell death by apoptosis, as did overexpression of this dominant-negative mutant. The data indicate that Repo-Man forms an essential complex with PP1γ and is required for the recruitment of PP1 to chromatin.

Evolution of protein phosphatases in plants and animals.

Protein phosphorylation appears to be a universal mechanism of protein regulation. Genomics has provided the means to compile inventories of protein phosphatases across a wide selection of organisms and this has supplied insights into the evolution of this group of enzymes. Protein phosphatases evolved independently several times yielding the groups we observe today. Starting from a core catalytic domain, phosphatases evolved by a series of gene duplication events and by adopting the use of regulatory subunits and/or fusion with novel functional modules or domains. Recent analyses also suggest that the serine/threonine specific enzymes are more ancient than the PTPs (protein tyrosine phosphatases). It is likely that the latter played a key role at the onset of metazoan evolution in conjunction with the tremendous expansion of tyrosine kinases and PTPs at this point. In the present review, we discuss the evolution of the PTPs, the serine/threonine specific PPP (phosphoprotein phosphatase) and PPM (metallo-dependent protein phosphatase) families and the more recently discovered phosphatases that utilize an aspartate-based catalytic mechanism. We will also highlight examples of convergent evolution and several phosphatases which are unique to plants.

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR.

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.

Large-scale Identification of Tubulin-binding Proteins Provides Insight on Subcellular Trafficking, Metabolic Channeling, and Signaling in Plant Cells

Microtubules play an essential role in the growth and development of plants and are known to be involved in regulating many cellular processes ranging from translation to signaling. In this article, we describe the proteomic characterization of Arabidopsis tubulin-binding proteins that were purified using tubulin affinity chromatography. Microtubule co-sedimentation assays indicated that most, if not all, of the proteins in the tubulin-binding protein fraction possessed microtubule-binding activity. Two-dimensional gel electrophoresis of the tubulin-binding protein fraction was performed, and 86 protein spots were excised and analyzed for protein identification. A total of 122 proteins were identified with high confidence using LC-MS/MS. These proteins were grouped into six categories based on their predicted functions: microtubule-associated proteins, translation factors, RNA-binding proteins, signaling proteins, metabolic enzymes, and proteins with other functions. Almost one-half of the proteins identified in this fraction were related to proteins that have previously been reported to interact with microtubules. This study represents the first large-scale proteomic identification of eukaryotic cytoskeleton-binding proteins, and provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells.

Protein Phosphatase 6 Interacts with the DNA-Dependent Protein Kinase Catalytic Subunit and Dephosphorylates γ-H2AX

ABSTRACT The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G2/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (γ-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of γ-H2AX, the dissolution of IR-induced foci, and release from the G2/M checkpoint in vivo.

Evolutionary Radiation Pattern of Novel Protein Phosphatases Revealed by Analysis of Protein Data from the Completely Sequenced Genomes of Humans, Green Algae, and Higher Plants

In addition to the major serine/threonine-specific phosphoprotein phosphatase, Mg2+-dependent phosphoprotein phosphatase, and protein tyrosine phosphatase families, there are novel protein phosphatases, including enzymes with aspartic acid-based catalysis and subfamilies of protein tyrosine phosphatases, whose evolutionary history and representation in plants is poorly characterized. We have searched the protein data sets encoded by the well-finished nuclear genomes of the higher plants Arabidopsis (Arabidopsis thaliana) and Oryza sativa, and the latest draft data sets from the tree Populus trichocarpa and the green algae Chlamydomonas reinhardtii and Ostreococcus tauri, for homologs to several classes of novel protein phosphatases. The Arabidopsis proteins, in combination with previously published data, provide a complete inventory of known types of protein phosphatases in this organism. Phylogenetic analysis of these proteins reveals a pattern of evolution where a diverse set of protein phosphatases was present early in the history of eukaryotes, and the division of plant and animal evolution resulted in two distinct sets of protein phosphatases. The green algae occupy an intermediate position, and show similarity to both plants and animals, depending on the protein. Of specific interest are the lack of cell division cycle (CDC) phosphatases CDC25 and CDC14, and the seeming adaptation of CDC14 as a protein interaction domain in higher plants. In addition, there is a dramatic increase in proteins containing RNA polymerase C-terminal domain phosphatase-like catalytic domains in the higher plants. Expression analysis of Arabidopsis phosphatase genes differentially amplified in plants (specifically the C-terminal domain phosphatase-like phosphatases) shows patterns of tissue-specific expression with a statistically significant number of correlated genes encoding putative signal transduction proteins.

The PII Signal Transduction Protein of Arabidopsis thaliana Forms an Arginine-regulated Complex with Plastid N-Acetyl Glutamate Kinase

The PII proteins are key mediators of the cellular response to carbon and nitrogen status and are found in all domains of life. In eukaryotes, PII has only been identified in red algae and plants, and in these organisms, PII localizes to the plastid. PII proteins perform their role by assessing cellular carbon, nitrogen, and energy status and conferring this information to other proteins through proteinprotein interaction. We have used affinity chromatography and mass spectrometry to identify the PII-binding proteins of Arabidopsis thaliana. The major PII-interacting protein is the chloroplast-localized enzyme N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. The interaction of PII with N-acetyl glutamate kinase was confirmed through pull-down, gel filtration, and isothermal titration calorimetry experiments, and binding was shown to be enhanced in the presence of the downstream product, arginine. Enzyme kinetic analysis showed that PII increases N-acetyl glutamate kinase activity slightly, but the primary function of binding is to relieve inhibition of enzyme activity by the pathway product, arginine. Knowing the identity of PII-binding proteins across a spectrum of photosynthetic and non-photosynthetic organisms provides a framework for a more complete understanding of the function of this highly conserved signaling protein.