A homologue of the human STRIPAK complex controls sexual development in fungi

Sexual development in fungi is a complex process involving the generation of new cell types and tissues - an essential step for all eukaryotic life. The characterization of sterile mutants in the ascomycete Sordaria macrospora has led to a number of proteins involved in sexual development, but a link between these proteins is still missing. Using a combined tandem-affinity purification/mass spectrometry approach, we showed in vivo association of developmental protein PRO22 with PRO11, homologue of mammalian striatin, and SmPP2AA, scaffolding subunit of protein phosphatase 2A. Further experiments extended the protein network to the putative kinase activator SmMOB3, known to be involved in sexual development. Extensive yeast two-hybrid studies allowed us to pinpoint functional domains involved in protein-protein interaction. We show for the first time that a number of already known factors together with new components associate in vivo to form a highly conserved multi-subunit complex. Strikingly, a similar complex has been described in humans, but the function of this so-called striatin interacting phosphatase and kinase (STRIPAK) complex is largely unknown. In S. macrospora, truncation of PRO11 and PRO22 leads to distinct defects in sexual development and cell fusion, indicating a role for the fungal STRIPAK complex in both processes.     

Large-scale analysis of phosphorylated proteins in maize leaf

Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation.     

The STRIPAK complex orchestrates cell wall integrity signalling to govern the fungal development and virulence of Fusarium graminearum

Striatin-interacting phosphatases and kinases (STRIPAKs) are evolutionarily conserved supramolecular complexes that control various important cellular processes such as signal transduction and development. However, the role of the STRIPAK complex in pathogenic fungi remains elusive. In this study, the components and function of the STRIPAK complex were investigated in Fusarium graminearum, an important plant-pathogenic fungus. The results obtained from bioinformatic analyses and the protein–protein interactome suggested that the fungal STRIPAK complex consisted of six proteins: Ham2, Ham3, Ham4, PP2Aa, Ppg1, and Mob3. Deletion mutations of individual components of the STRIPAK complex were created, and observed to cause a significant reduction in fungal vegetative growth and sexual development, and dramatically attenuae virulence, excluding the essential gene PP2Aa. Further results revealed that the STRIPAK complex interacted with the mitogen-activated protein kinase Mgv1, a key component in the cell wall integrity pathway, subsequently regulating the phosphorylation level and nuclear accumulation of Mgv1 to control the fungal stress response and virulence. Our results also suggested that the STRIPAK complex was interconnected with the target of rapamycin pathway through Tap42-PP2A cascade. Taken together, our findings revealed that the STRIPAK complex orchestrates cell wall integrity signalling to govern the fungal development and virulence of F. graminearum and highlighted the importance of the STRIPAK complex in fungal virulence.     

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.     

The STRIPAK component SipC is involved in morphology and cell-fate determination in the nematode-trapping fungus Duddingtonia flagrans

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a highly conserved eukaryotic signaling hub involved in the regulation of many cellular processes. In filamentous fungi, STRIPAK controls multicellular development, hyphal fusion, septation, and pathogenicity. In this study, we analyzed the role of the STRIPAK complex in the nematode-trapping fungus Duddingtonia flagrans which forms three-dimensional, adhesive trapping networks to capture Caenorhabditis elegans. Trap networks consist of several hyphal loops which are morphologically and functionally different from vegetative hyphae. We show that lack of the STRIPAK component SipC (STRIP1/2/HAM-2/PRO22) results in incomplete loop formation and column-like trap structures with elongated compartments. The misshapen or incomplete traps lost their trap identity and continued growth as vegetative hyphae. The same effect was observed in the presence of the actin cytoskeleton drug cytochalasin A. These results could suggest a link between actin and STRIPAK complex functions.     

HAM‐2 and HAM‐3 are central for the assembly of the Neurospora STRIPAK complex at the nuclear envelope and regulate nuclear accumulation of the MAP kinase MAK‐1 in a MAK‐2‐dependent manner

Intercellular communication and somatic cell fusion are important for fungal colony establishment, multicellular differentiation and have been associated with host colonization and virulence of pathogenic species. By a combination of genetic, biochemical and live cell imaging techniques, we characterized the Neurospora crassa STRIPAK complex that is essential for self‐signalling and consists of the six proteins HAM‐2/STRIP, HAM‐3/striatin, HAM‐4/SLMAP, MOB‐3/phocein, PPG‐1/PP2A‐C and PP2A‐A. We describe that the core STRIPAK components HAM‐2 and HAM‐3 are central for the assembly of the complex at the nuclear envelope, while the phosphatase PPG‐1 only transiently associates with this central subcomplex. Our data connect the STRIPAK complex with two MAP kinase pathways: (i) nuclear accumulation of the cell wall integrity MAP kinase MAK‐1 depends on the functional integrity of the STRIPAK complex at the nuclear envelope, and (ii) phosphorylation of MOB‐3 by the MAP kinase MAK‐2 impacts the nuclear accumulation of MAK‐1. In summary, these data support a model, in which MAK‐2‐dependent phosphorylation of MOB‐3 is part of a MAK‐1 import mechanism. Although self‐communication remained intact in the absence of nuclear MAK‐1 accumulation, supporting the presence of multiple mechanisms that co‐ordinate robust intercellular communication, proper fruiting body morphology was dependent on the MAK‐2‐phosphorylated N‐terminus of MOB‐3.     

Phosphoproteomics strategies for the functional analysis of signal transduction

Protein phosphorylation is a key regulatory mechanism of cellular signalling processes. The analysis of phosphorylated proteins and the characterisation of phosphorylation sites under different biological conditions are some of the most challenging tasks in current proteomics research. Reduction of the sample complexity is one major step for the analysis of low-abundance kinase substrates, which can be achieved by various subcellular fractionation techniques. One strategy is the enrichment of phosphorylated proteins or peptides by immunoprecipitation or chromatography, e.g. immobilised metal affinity chromatography, prior to analysis. 2-DE gels are powerful tools for the analysis of phosphoproteins when combined with new multiplexing techniques like DIGE, phosphospecific stains, autoradiography or immunoblotting. In addition, several gel-free methods combining chromatography with highly sensitive MS have been successfully applied for the analysis of complex phosphoproteomes. Recently developed approaches like KESTREL or 'chemical genetics' and also protein microarrays offer new possibilities for the identification of specific kinase targets. This review summarises various strategies for the analyses of phosphoproteins with a special focus on the identification of novel kinase substrates.     

Striatins Contain a Noncanonical Coiled Coil That Binds Protein Phosphatase 2A A Subunit to Form a 2:2 Heterotetrameric Core of Striatin-interacting Phosphatase and Kinase (STRIPAK) Complex

The protein phosphatase 2A (PP2A) and kinases such as germinal center kinase III (GCKIII) can interact with striatins to form a supramolecular complex called striatin-interacting phosphatase and kinase (STRIPAK) complex. Despite the fact that the STRIPAK complex regulates multiple cellular events, it remains only partially understood how this complex itself is assembled and regulated for differential biological functions. Our recent work revealed the activation mechanism of GCKIIIs by MO25, as well as how GCKIIIs heterodimerize with CCM3, a molecular bridge between GCKIII and striatins. Here we dissect the structural features of the coiled coil domain of striatin 3, a novel type of PP2A regulatory subunit that functions as a scaffold for the assembly of the STRIPAK complex. We have determined the crystal structure of a selenomethionine-labeled striatin 3 coiled coil domain, which shows it to assume a parallel dimeric but asymmetric conformation containing a large bend. This result combined with a number of biophysical analyses provide evidence that the coiled coil domain of striatin 3 and the PP2A A subunit form a stable core complex with a 2:2 stoichiometry. Structure-based mutational studies reveal that homodimerization of striatin 3 is essential for its interaction with PP2A and therefore assembly of the STRIPAK complex. Wild-type striatin 3 but not the mutants defective in PP2A binding strongly suppresses apoptosis of Jurkat cells induced by the GCKIII kinase MST3, most likely through a mechanism in which striatin recruits PP2A to negatively regulate the activation of MST3. Collectively, our work provides structural insights into the organization of the STRIPAK complex and will facilitate further functional studies.     

Role of Cdc42-Cla4 interaction in the pheromone response of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the highly conserved Rho-type GTPase Cdc42 is essential for cell division and controls cellular development during mating and invasive growth. The role of Cdc42 in mating has been controversial, but a number of previous studies suggest that the GTPase controls the mitogen-activated protein (MAP) kinase cascade by activating the p21-activated protein kinase (PAK) Ste20. To further explore the role of Cdc42 in pheromone-stimulated signaling, we isolated novel alleles of CDC42 that confer resistance to pheromone. We find that in CDC42(V36A) and CDC42(V36A, I182T) mutant strains, the inability to undergo pheromone-induced cell cycle arrest correlates with reduced phosphorylation of the mating MAP kinases Fus3 and Kss1 and with a decrease in mating efficiency. Furthermore, Cdc42(V36A) and Cdc42(V36A, I182T) proteins show reduced interaction with the PAK Cla4 but not with Ste20. We also show that deletion of CLA4 in a CDC42(V36A, I182T) mutant strain suppresses pheromone resistance and that overexpression of CLA4 interferes with pheromone-induced cell cycle arrest and MAP kinase phosphorylation in CDC42 wild-type strains. Our data indicate that Cla4 has the potential to act as a negative regulator of the mating pathway and that this function of the PAK might be under control of Cdc42. In conclusion, our study suggests that control of pheromone signaling by Cdc42 not only depends on Ste20 but also involves interaction of the GTPase with Cla4.     

Drosophila Lk6 kinase controls phosphorylation of eukaryotic translation initiation factor 4E and promotes normal growth and development

Eukaryotic initiation factor 4E (eIF4E) controls a crucial step of translation initiation and is critical for cell growth . Biochemical studies have shown that it undergoes a regulated phosphorylation by the MAP-kinase signal-integrating kinases Mnk1 and Mnk2 . Although the role of eIF4E phosphorylation in mammalian cells has remained elusive , recent work in Drosophila has established that it is required for growth and development . Here, we demonstrate that a previously identified Drosophila kinase called Lk6 is the functional homolog of mammalian Mnk kinases. We generated lk6 loss-of-function alleles and found that eIF4E phosphorylation is dramatically reduced in lk6 mutants. Importantly, lk6 mutants exhibit reduced viability, slower development, and reduced adult size, demonstrating that Lk6 function is required for organismal growth. Moreover, we show that uniform lk6 expression rescues the lethality of eIF4E hypomorphic mutants in an eIF4E phosphorylation site-dependent manner and that the two proteins participate in a common complex in Drosophila S2 cells, confirming the functional link between Lk6 and eIF4E. This work demonstrates that Lk6 exerts a tight control on eIF4E phosphorylation and is necessary for normal growth and development.     

Large-scale analysis of protein phosphorylation in Populus leaves

Protein phosphorylation is a key regulatory factor in all aspects of plant biology; most regulatory pathways are governed by the reversible phosphorylation of proteins. To better understand the role that phosphorylated proteins play in a woody model plant, we performed a systemic analysis of the phosphoproteome from Populus leaves using high accuracy NanoLC–MS/MS in combination with biochemical enrichments using strong cation exchange chromatography and titanium dioxide chromatography. We identified 104 phosphopeptides from 94 phosphoproteins and determined 111 phosphorylation sites including 93 occurring on serine residues and 18 on threonine residues. The identified phosphoproteins are involved in a wide variety of metabolic processes. Among these identified phosphoproteins, 68 phosphorylation sites (72 %) were located outside of conserved domains. The identified phosphopeptides share a common phosphorylation motif of pS/pT-P/D-S/A. These data suggest that the Populus metabolism and gene regulation machinery are major targets of phosphorylation. To our knowledge, this is the first gel-free, large-scale phosphoproteomics analysis in woody plants. The identified phosphorylation sites will be a valuable resource for many fields of plant biology, and information gained from the study will provide a better understanding of protein phosphorylation.     

Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.     

Functional Analysis of Centrosomal Kinase Substrates in Drosophila melanogaster Reveals a New Function of the Nuclear Envelope Component Otefin in Cell Cycle Progression

Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIβ. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.     

Deletion of Smgpi1 encoding a GPI‐anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora

The str iatin i nteracting p hosphatase a nd k inase (STRIPAK) complex, which is composed of striatin, protein phosphatase PP2A and kinases, is required for fruiting‐body development and cell fusion in the filamentous ascomycete Sordaria macrospora. Here, we report on the interplay of the g lycosylp hosphatidyli nositol (GPI)‐anchored protein SmGPI1 with the kinase activator SmMOB3, a core component of human and fungal STRIPAK complexes. SmGPI1 is conserved among filamentous ascomycetes and was first identified in a yeast two‐hybrid screen using SmMOB3 as bait. The physical interaction of SmMOB3 and SmGPI1 was verified by co‐immunoprecipitation. In vivo localization and differential centrifugation revealed that SmGPI1 is predominantly secreted and attached to the cell wall but is also associated with mitochondria and appears to be a dual‐targeted protein. Deletion of Smgpi1 led to an increased number of fruiting bodies that were normally shaped but reduced in size. In addition, Smmob3 and Smgpi1 genetically interact. In the sterile ΔSmmob3 background deletion of Smgpi1 restores fertility, vegetative growth as well as hyphal‐fusion defects. The suppression effect was specific for the ΔSmmob3 mutant as deletion of Smgpi1 in other STRIPAK mutants does not restore fertility.     

The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.