Regulation of the stability and activity of CDC25A and CDC25B by protein phosphatase PP2A and 14-3-3 binding

Cyclin-dependent kinase (CDK)-activating phosphatases, CDC25A and CDC25B, are labile proteins, and their levels vary in a cell cycle-dependent manner. Immediate-early response IER5 protein negatively regulates the cellular CDC25B levels, and stress-induced IER5 expression potentiates G2/M arrest. IER5 binds to protein phosphatase PP2A and regulates the PP2A substrate specificity. We show that IER5 binds to CDC25B and assists PP2A to convert CDC25B to hypophosphorylated forms. Hypophosphorylation at Ser323 results in the dissociation of CDC25B from 14‐3-3 phospho-binding proteins. In IER5 expressing cells, CDC25B dissociated from 14‐3-3 is unstable but slightly activated, because 14‐3-3 inhibits CDC25B polyubiquitination and CDC25B binding to CDK1. The 14‐3-3 binding to CDC25A also impedes CDC25A degradation and CDC25A-CDK2 interaction. We propose that 14‐3-3 is an important regulator of CDC25A and CDC25B and that PP2A/IER5 controls the stability and activity of CDC25B through regulating the interaction of CDC25B and 14‐3-3.     

Regulation of Endosome Sorting by a Specific PP2A Isoform

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869–5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205–216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.     

NIPP1-mediated interaction of protein phosphatase-1 with CDC5L, a regulator of pre-mRNA splicing and mitotic entry

NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles. We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module. A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G(2)/M progression and pre-mRNA splicing. CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells. Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments. The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g. by cyclin E-Cdk2. When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G(2)/M transition. However, the FHA domain blocked beta-globin pre-mRNA splicing in nuclear extracts. A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects. We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.     

Leucine methylation of protein phosphatase PP4C at C-terminal is critical for its cellular functions

Background

Protein phosphatase 4 (PP4) has been known to have critical functions in DNA double strand break (DSB) repair and cell cycle by the regulation of phosphorylation of its target proteins, such as H2AX, RPA2, KAP-1, 53BP1. However, it is largely unknown how PP4 itself is regulated.

Methods

We examined the PP4C methylation on L307 at C-terminal by using methylated-leucine specific antibody. Then with PP4C L307A mutant, we explored that how nonmethylated form of PP4C affects its known cellular functions by immunoprecipitation, immunofluorescence, and DNA DSB repair assays.

Results

Here we show that PP4C is methylated on its C-terminal leucine residue in vivo and this methylation is important for cellular functions mediated by PP4. In the cells PP4C L307A mutant has significantly low activity of dephosphorylation against its known target proteins, and the loss of interaction between L307A PP4 mutant and regulatory subunits, R1, R2, or R3α/β causes the dissociation from its target proteins. Moreover, PP4C L307A mutant loses its role in both DSB repair pathways, HR (homologous recombination) and NHEJ (non-homologous end joining), which phenocopies PP4C depletion.

Conclusion

Our results demonstrate the key site of PP4C methylation and establish the physiological importance of this regulation.     

S100 proteins modulate protein phosphatase 5 function: a link between CA2+ signal transduction and protein dephosphorylation

PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca(2+)/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca(2+)-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction.     

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca²⁺/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca²⁺/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca²⁺/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca²⁺/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca²⁺-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.     

Functional divergence of Brassica napus BnaABI1 paralogs in the structurally conserved PP2CA gene subfamily of Brassicaceae

Group A PP2C (PP2CA) genes form a gene subfamily whose members play an important role in regulating many biological processes by dephosphorylation of target proteins. In this study we examined the effects of evolutionary changes responsible for functional divergence of BnaABI1 paralogs in Brassica napus against the background of the conserved PP2CA gene subfamily in Brassicaceae. We performed comprehensive phylogenetic analyses of 192 PP2CA genes in 15 species in combination with protein structure homology modeling. Fundamentally, the number of PP2CA genes remained relatively constant in these taxa, except in the Brassica genus and Camelina sativa. The expansion of this gene subfamily in these species has resulted from whole genome duplication. We demonstrated a high degree of structural conservation of the PP2CA genes, with a few minor variations between the different PP2CA groups. Furthermore, the pattern of conserved sequence motifs in the PP2CA proteins and their secondary and 3D structures revealed strong conservation of the key ion-binding sites. Syntenic analysis of triplicated regions including ABI1 paralogs revealed significant structural rearrangements of the Brassica genomes. The functional and syntenic data clearly show that triplication of BnaABI1 in B. napus has had an impact on its functions, as well as the positions of adjacent genes in the corresponding chromosomal regions. The expression profiling of BnaABI1 genes showed functional divergence, i.e. subfunctionalization, potentially leading to neofunctionalization. These differences in expression are likely due to changes in the promoters of the BnaABI1 paralogs. Our results highlight the complexity of PP2CA gene subfamily evolution in Brassicaceae.     

Low temperature-stimulated phosphorylation regulates the binding of nuclear factors to the promoter of Wcs120, a cold-specific gene in wheat

The Wcs120 gene encodes a highly abundant protein which appears to play an important role during cold acclimation of wheat. To understand the regulatory mechanism controlling its expression at low temperature, the promoter region has been characterized. Electrophoretic mobility shift assays using short promoter fragments revealed the presence in nuclear extracts from non-acclimated (NA) plants of multiple DNA-binding proteins which interact with several elements. In contrast, no DNA-binding activity was observed in the nuclear extracts from cold-acclimated (CA) plants. In vitro dephosphorylation of these CA nuclear extracts with alkaline phosphatase restored the binding activity. Moreover, okadaic acid (a potent phosphatase inhibitor) markedly stimulated the in vivo accumulation of the WCS120 family of proteins. This suggests that protein phosphatases PP1 and/or PP2A negatively regulate the expression of the Wcs120 gene. In addition, both Ca²⁺-dependent and Ca²⁺-independent kinase activities were found to be significantly higher in the CA nuclear extracts. Western analysis using antibodies directed against protein kinase C (PKC) isoforms showed that a PKCγ homolog (84?kDa) is selectively translocated into the nucleus in response to low temperature. Taken together, our results suggest that, in vivo, the expression of the Wcs120 gene may be regulated by nuclear factors whose binding activity is modulated by a phosphorylation/dephosphorylation mechanism.     

V‐ATPase deactivation in blowfly salivary glands is mediated by protein phosphatase 2C

The activity of vacuolar H⁺‐ATPase (V‐ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5‐HT). 5‐HT induces, via protein kinase A, the phosphorylation of V‐ATPase subunit C and the assembly of V‐ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V‐ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK‐506) do not prevent V‐ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg²⁺ level caused by loading secretory cells with EDTA‐AM leads to the activation of proton pumping in the absence of 5‐HT, prolongs the 5‐HT‐induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V‐ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg²⁺, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.     

Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation

The B″/PR72 family of protein phosphatase 2A (PP2A) is an important PP2A family involved in diverse cellular processes, and uniquely regulated by calcium binding to the regulatory subunit. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here, we report crystal structures of the isolated PR72 and the trimeric PR70 holoenzyme at a resolution of 2.1 and 2.4 Å, respectively, and in vitro characterization of Cdc6 dephosphorylation. The holoenzyme structure reveals that one of the PR70 calcium-binding motifs directly contacts the scaffold subunit, resulting in the most compact scaffold subunit conformation among all PP2A holoenzymes. PR70 also binds distinctively to the catalytic subunit near the active site, which is required for PR70 to enhance phosphatase activity toward Cdc6. Our studies provide a structural basis for unique regulation of B″/PR72 holoenzymes by calcium ions, and suggest the mechanisms for precise control of substrate specificity among PP2A holoenzymes.     

A cyclophilin-regulated PP2A-like protein phosphatase in thylakoid membranes of plant chloroplasts.

Dephosphorylation of central photosynthetic proteins regulates their turnover in plant thylakoid membranes. A membrane protein phosphatase from spinach thylakoids was purified 13000-fold using detergent-engaged FPLC. The purified enzyme exhibited characteristics typical of eukaryotic Ser/Thr phosphatases of the PP2A family in that it was inhibited by okadaic acid (IC(50) = 0.4 nM) and tautomycin (IC(50) = 25 nM), irreversibly bound to microcystin-agarose, and recognized by a polyclonal antibody raised against a recombinant catalytic subunit of human PP2A. Furthermore, the anti-PP2A antibody inhibited protein dephosphorylation in isolated thylakoids. The phosphatase copurified with TLP40, a cyclophilin-like peptidyl-prolyl isomerase located in the thylakoid lumen. TLP40 could be released from the phosphatase immobilized on microcystin-agarose by high-salt treatment. Binding of cyclosporin A (CsA) to TLP40 led to thylakoid phosphatase activation, while cyclophilin substrates, prolyl-containing oligopeptides, inhibited protein dephosphorylation. This dephosphorylation could be modulated by CsA or oligopeptides only after the thylakoids had been ruptured to expose the lumenal membrane surface where the TLP40 is located. Regulation of the PP2A-like phosphatase at the outer thylakoid surface is likely to operate via reversible binding of TLP40 to the inner membrane surface. This is a first example of transmembrane regulation in which the activity of phosphatase is altered by the binding of a cyclophilin to a site other than the active one. We propose that signaling from TLP40 to the protein phosphatase coordinates dephosphorylation and protein folding, two processes required for protein turnover during the repair of photoinhibited photosystem II reaction centers.     

Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1

CDC5L and PLRG1 are both spliceosomal proteins that are highly conserved across species. They have both been shown to be part of sub- spliceosomal protein complexes that are essential for pre-mRNA splicing in yeast and humans. CDC5L and PLRG1 interact directly in vitro. This interaction is mediated by WD40 regions in PLRG1 and the C-terminal domain of CDC5L. In order to determine whether this interaction is important for the splicing mechanism, we have designed peptides corresponding to highly conserved sequences in the interaction domains of both proteins. These peptides were used in in vitro splicing experiments as competitors to the cognate sequences in the endogenous proteins. Certain peptides derived from the binding domains of both proteins were found to inhibit in vitro splicing. This splicing inhibition could be prevented by preincubating the peptides with the corresponding partner protein that had been expressed in Escherichia coli. The results from this study indicate that the interaction between CDC5L and PLRG1 is essential for pre-mRNA splicing and further demonstrate that small peptides can be used as effective splicing inhibitors.     

Two subclasses of guanine exchange factor (GEF) domains revealed by comparison of activities of chimeric genes constructed from CDC25, SDC25 and BUD5 in Saccharomyces cerevisiae

Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes. We have assessed the structure-function relationships between three different members of this family in S. cerevisiae, Cdc25p, Sdc25p and Bud5p. Cdc25p controls the Ras pathway, whereas Bud5p controls bud site localization. We demonstrate that the GEF domain of Sdc25p is closely related to that of Cdc25p. We first constructed a thermosensitive allele of SDC25 by specifically altering amino acid positions known to be changed in the cdc25-1 mutation. Secondly, we constructed three chimeric genes from CDC25 and SDC25, the products of which are as active in the Ras pathway as are the wild-type proteins. In contrast, similar chimeras made between CDC25 and BUD5 lead to proteins that are inactive both in the Ras and budding control pathways. This difference in the ability of chimeric proteins to retain activity allows us to define two subclasses of structurally different GEFs: Cdc25p and Sdc25p are Ras-specific GEFs, and Bud5p is a putative GEF for the Rsr1/Bud1 Rap-like protein.