Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry

Functional proteomics provides a powerful method for monitoring global molecular responses following activation of signal transduction pathways, reporting altered protein posttranslational modification and expression. Here we combine functional proteomics with selective activation and inhibition of MKK1/2, in order to identify cellular targets regulated by the MKK/ERK cascade. Twenty-five targets of this signaling pathway were identified, of which only five were previously characterized as MKK/ERK effectors. The remaining targets suggest novel roles for this signaling cascade in cellular processes of nuclear transport, nucleotide excision repair, nucleosome assembly, membrane trafficking, and cytoskeletal regulation. This study represents an application of functional proteomics toward identifying regulated targets of a discrete signal transduction pathway and demonstrates the utility of this discovery-based strategy in elucidating novel MAP kinase pathway effectors.     

Regulation of Hepatitis C virus replication and gene expression by the MAPK-ERK pathway

The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation of multiple cellular processes including the cell cycle. In the present study using a Huh7 cell line Con1 with an HCV replicon, we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-α signalling. Epithelial growth factor (EGF) was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Con1 cells. It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site. Consistently, a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfection assays. Thus, the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication. In addition, cyclin-dependent kinases (CDKs) downstream of ERK may also be involved in the modulation of HCV replication since roscovitine, an inhibitor of CDKs had a similar effect to that of U0126. Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels. Further, the replication of HCV replicon in Con1 cells was inhibited by IFN-α. The inhibitory effect of IFN-α could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs. It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.     

Identification of a novel Ser/Thr protein phosphatase Ppq1 as a negative regulator of mating MAP kinase pathway in Saccharomyces cerevisiae

The specificity and efficiency of cell signaling is largely governed by the complex formation of signaling proteins. The precise spatio-temporal control of the complex assembly is crucial for proper signaling and cell survival. Protein phosphorylation is a key mechanism of signal processing in most of cell signaling networks. Phosphatases, along with kinases, control the phosphorylation state of many proteins and thus play a critical role in the precise regulation of signaling at each stage such as activation, propagation, and adaptation. Identification and functional analysis of pathway-specific phosphatase is, therefore, crucial for the understanding of cell signaling mechanisms. Here, we have developed a novel screening strategy to identify pathway-specific phosphatases, in which the entire repertoire of cell’s phosphatases was tethered to a signaling complex and the changes in signaling response were monitored. As a model target, we have chosen the mating MAP kinase pathway in the budding yeast, which is composed of three kinases and Ste5 scaffold protein. Using this strategy, a putative Ser/Thr phosphatase, Ppq1, was identified to be mating-specific. Results show that Ppq1 down-regulates mating signaling by targeting at or upstream of the terminal MAP kinase Fus3 in the cascade. The catalytic activity of Ppq1 as a phosphatase was confirmed in vitro and is necessary for its function in the regulation of mating signaling. Overall, the data suggest that Ppq1 functions as a negative regulator of mating MAPK pathway by dephosphorylating target pathway protein(s) and plays a key role in the control of the background signaling noise.     

Differential activation of ERK1,2 MAP kinase signaling pathway in mesenchymal stem cell from control and osteoporotic postmenopausal women

Osteoblasts, the cells responsible for bone formation, derive from mesenchymal stem cells (MSCs) in bone marrow. To acquire a new cell phenotype, uncommitted MSCs must undergo several proliferation and differentiation changes. Although, it is known that extracellular signal-regulated protein kinases (ERKs) mitogen-activated protein (MAP) kinase pathway signaling is involved in the proliferation and differentiation processes, the role of ERKs in osteogenic differentiation it is controversial, at present. In addition, the function that ERK could play in MSCs derived from osteoporotic patients it is not well documented. In this study, we analyze whether previously observed differences in the dynamic response of MSCs from normal and osteoporotic postmenopausal women can be explained by changes in the activation of this signal transduction pathway. Levels of ERK phosphorylation and their correlation with osteogenic differentiation were evaluated in cultures of MSCs derived from osteoporotic postmenopausal women and "healthy" controls. The results show that, under basal conditions, MSCs derived from osteoporotic donors show a level of ERK phosphorylation 2.5 times higher than MSCs derived from control donors. The addition of the osteogenic stimulus only slightly increases the p-ERK level in cells derived from osteoporotic donors, and is higher in cells derived from control women. Important differences in the ability of PD98059 to inhibit phosphorylation of ERK in both types of cells were also observed, as well as the effect that this inhibition produced on calcium deposition. We conclude that the MAP kinase pathway signaling is differentially activated in MSCs derived from osteoporotic postmenopausal women. The high p-ERK levels in MSC derived from osteoporotic donors could determine the unresponsiveness of these cells to the osteogenic differentiation stimulus.     

The p53 pathway is synergized by p38 MAPK signaling to mediate 11,11'-dideoxyverticillin-induced G2/M arrest

The phytochemical 11,11'-dideoxyverticillin, derived from the fungus Shiraia bambusicola, has been shown to possess potent anticancer activity in vitro and in vivo. Here, we investigated the effect of 11,11'-dideoxyverticillin on cell cycle progression, and explored the potential mechanisms for this effect. A concentration- and time-dependent cell cycle blockade at G2/M phase was observed in human colon cancer cells (HCT-116) following 11,11'-dideoxyverticillin treatment and was associated with marked increases in levels of p53, phospho-p53(ser20) and phospho-Chk2(Thr 68). When wild type p53 expression was specifically inhibited by RNA interference, HCT-116 cells treated with 11,11'-dideoxyverticillin failed to arrest in G2/M and did not show increased phospho-Chk2(Thr 68). On the other hand, 11,11'-dideoxyverticillin treatment also elicited p38 MAP kinase activity and expression of phospho-p38 MAPK. Treatment with a specific p38 MAPK inhibitor (SB203580) successfully inhibited p38 MAPK and delayed the onset of G2/M arrest induced by 0.5 microM 11,11'-dideoxyverticillin after approximately 6 h, but did not abolish the induction of G2/M arrest. Additionally, SB203580 did not alter the levels of p53, phospho-p53 (ser20), or phospho-Chk2 (Thr68) proteins in 11,11'-dideoxyverticillin-treated cells. Together, these findings indicate that p53-mediated phosphorylation of Chk2 maybe plays a vital role in 11,11'-dideoxyverticillin-induced G2/M arrest, and that p38 MAPK might accelerate this progression. Our work suggests a new possibility of interactions among p53, Chk2 and p38 MAPK signaling in G2/M arrest.     

QIKS – Quantitative identification of kinase substrates

Signaling networks regulate cellular responses to external stimuli through post‐translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large‐scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge. To address this problem, we present a novel approach for the identification of direct kinase substrates using chemical genetics in combination with quantitative phosphoproteomics. Quantitative identification of kinase substrates (QIKS) is a novel‐screening platform developed for the proteome‐wide substrate‐analysis of specific kinases. Here, we aimed to identify substrates of mitogen‐activated protein kinase/Erk kinase (Mek1), an essential kinase in the mitogen‐activated protein kinase cascade. An ATP analog‐sensitive mutant of Mek1 (Mek1‐as) was incubated with a cell extract from Mek1 deficient cells. Phosphorylated proteins were analyzed by LC‐MS/MS of IMAC‐enriched phosphopeptides, labeled differentially for relative quantification. The identification of extracellular regulated kinase 1/2 as the sole cytoplasmic substrates of MEK1 validates the applicability of this approach and suggests that QIKS could be used to identify substrates of a wide variety of kinases.     

Relevance of histone H1 kinase activity to the G2/M transition during the cell cycle ofDictyostelium discoideum

The implication of histone H1 kinase activity for the G2/M transition during the cell cycle was investigated usingDictyostelium discoideum Ax-2. Histone H1 kinase with its activity was purified from cell extracts by the use of p13^suc1 affinity gel. In the vegetative cell cycle, the activity of histone H1 kinase including Cdc2 kinase was found using synchronized Ax-2 cells to be highest just before the entry into mitosis. The activity also was markedly enhanced just prior to the M phase from which developing cells (possibly prespore cells) reinitiate their cell cycle at the mound-tipped aggregate stage. These results strongly suggest the importance of Cdc2 kinase activity in the G2 to M phase transition during the cell cycle, as the case for other eukaryotic cells.     

Activation of mitogen activated protein (MAP) kinases by vanadate is independent of insulin receptor autophosphorylation

Treatment of Chinese hamster ovary (CHO) cells over-expressing the human insulin receptor (CHO-HIRc) with the insulin mimetic agent, vanadate, resulted in a dose- and time-dependent tyrosine phosphorylation of two proteins with apparent molecular sizes of 42 kDa (p42) and 44 kDa (p44). However, vanadate was unable to stimulate the tyrosyi phosphorylation of theβ-subunit of the insulin receptor. By using myelin basic protein (MBP) as the substrate to measure mitogen-activated protein (MAP) kinase activity in whole cell lysates, vanadate-stimulated tyrosyl phosphorylation of p42 and p44 was associated with a dose- and time-dependent activation of MAP kinase activity. Furthermore, affinity purification of cell lysates on anti-phosphotyrosine agarose column followed by immunoblotting with a specific antibody to MAP kinases demonstrated that vanadate treatment increased the tyrosyl phosphorylation of both p44^mapk and p42^mapk by several folds, as compared to controls, in concert with MAP kinase activation. In addition, retardation in gel mobility further confirmed that vanadate treatment increased the phosphorylation of p44^mapk and p42^mapk in CHO-HIRc. A similar effect of vanadate on MAP kinase tyrosyl phosphorylation and activation was also observed in CHO cells over-expressing a protein tyrosine kinase-deficient insulin receptor (CHO-1018). These results demonstrate that the protein tyrosine kinase activity of the insulin receptor may not be required in the signaling pathways leading to the vanadate-mediated tyrosyl phosphorylation and activation of MAP kinases.     

The multiple personalities of the regulatory subunit of protein kinase CK2: CK2 dependent and CK2 independent roles reveal a secret identity for CK2beta

Protein kinase CK2 (formerly casein kinase II), an enzyme that participates in a wide variety of cellular processes, has traditionally been classified as a stable tetrameric complex consisting of two catalytic CK2alpha or CK2alpha' subunits and two regulatory CK2beta subunits. While consideration of CK2 as a tetrameric complex remains relevant, significant evidence has emerged to challenge the view that its individual subunits exist exclusively within these complexes. This review will summarize biochemical and genetic evidence indicating that the regulatory CK2beta subunit exists and performs functions independently of CK2 tetramers. For example, unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumors. Furthermore, localization studies including live cell imaging have demonstrated that while the catalytic and regulatory subunits of CK2 exhibit extensive co-localization, independent mobility of the individual CK2 subunits can also be observed within cells. Identification of proteins that interact with CK2beta in the absence of catalytic CK2 subunits reinforces the notion that CK2beta has functions distinct from CK2 and begins to offer insights into these CK2-independent functions. In this respect, the discovery that CK2beta can interact with and modulate the activity of a number of other serine/threonine protein kinases including A-Raf, c-Mos and Chk1 is particularly striking. This review will discuss the interactions between CK2beta and these protein kinases with special emphasis on the properties of CK2beta that mediate these interactions and on the implications of these interactions in yielding new prospects for elucidation of the cellular functions of CK2beta.     

Cathelicidin stimulates colonic mucus synthesis by up-regulating MUC1 and MUC2 expression through a mitogen-activated protein kinase pathway

Mucus forms the physical barrier along the gastrointestinal tract. It plays an important role to prevent mucosal damage and inflammation. Our animal study showed that antibacterial peptide 'cathelicidin' increased mucus thickness and prevented inflammation in the colon. In the current study, we examined the direct effect and mechanisms by which the peptide increased mucus synthesis in a human colonic cell line (HT-29). Human cathelicidin (LL-37) dose-dependently (10-40 microg/ml) and significantly stimulated mucus synthesis by increasing the D-[6-(3)H] glucosamine incorporation in the cells. Real-time PCR data showed that addition of LL-37 induced more than 50% increase in MUC1 and MUC2 mRNA levels. Treatment with MUC1 and MUC2 siRNAs normalized the stimulatory action of LL-37 on mucus synthesis. LL-37 also activated the phosphorylation of mitogen-activated protein (MAP) kinase in the cells. A specific inhibitor of the MAP kinase pathway, U0126, completely blocked the increase of MUC1 and MUC2 expression as well as mucus synthesis by LL-37. Taken together, LL-37 can directly stimulate mucus synthesis through activation of MUC1 and MUC2 expression and MAP kinase pathway in human colonic cells.     

PD98059 and U0126 activate AMP-activated protein kinase by increasing the cellular AMP:ATP ratio and not via inhibition of the MAP kinase pathway

The MAP kinase pathway inhibitor U0126 caused phosphorylation and activation of AMP-activated protein kinase (AMPK) and increased phosphorylation of its downstream target acetyl-CoA carboxylase, in HEK293 cells. This effect only occurred in cells expressing the upstream kinase, LKB1. Of two other widely used MAP kinase pathway inhibitors not closely related in structure to U0126, PD98059 also activated AMPK but PD184352 did not. U0126 and PD98059, but not PD184352, also increased the cellular ADP:ATP and AMP:ATP ratios, accounting for their ability to activate AMPK. These results suggest the need for caution in interpreting experiments conducted using U0126 and PD98059.     

The mitogen-activated protein kinase p38 pathway is conserved in metazoans: cloning and activation of p38 of the SAPK2 subfamily from the sponge Suberites domuncula

Our recent data suggest that during auto- and allograft recognition in sponges (Porifera), cytokines are differentially expressed. Since the mitogen-activated protein kinase (MAPK) signal transduction modulates the synthesis and release of cytokines, we intended to identify one key molecule of this pathway. Therefore, a cDNA from the marine sponge Suberites domuncula encoding the MAPK was isolated and analyzed. Its encoded protein is 366 amino acids long (calculated Mr 42 209), has a TGY dual phosphorylation motif in protein kinase subdomain VIII and displays highest overall similarity to the mammalian p38 stress activated protein kinase (SAPK2), one subfamily of MAPKs. The sponge protein was therefore termed p38_SD. The overall homology (identity and similarity) between p38_SD and human p38alpha (CSBP2) kinase is 82%. One feature of the sponge kinase is the absence of threonine at position 106. In human p38alpha MAPK this residue is involved in the interaction with the specific pyridinyl-imidazole inhibitor; T106 is replaced in p38_SD by methionine. Inhibition studies with the respective inhibitor SB 203580 showed that it had no effect on the phosphorylation of the p38 substrate myelin basic protein. A stress responsive kinase Krs_SD similar to mammalian Ste20 kinases, upstream regulators of p38, had already previously been found in S. domuncula. The S. domuncula p38 MAPK is phosphorylated after treatment of the animal in hypertonic medium. In contrast, exposure of cells to hydrogen peroxide, heat shock and ultraviolet light does not cause any phosphorylation of p38. It is concluded that sponges, the oldest and most simple multicellular animals, utilize the conserved p38 MAPK signaling pathway, known to be involved in stress and immune (inflammatory) responses in higher animals.     

Retaining of the assembly capability of vimentin phosphorylated by mitogen-activated protein kinase-activated protein kinase-2

Intermediate filament (IF) networks can be regulated by phosphorylation of unit proteins, such as vimentin, by specific kinases leading to reorganization of the IF filamentous structure. Recently, we identified mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) as a vimentin kinase (Cheng and Lai [1998] J. Cell. Biochem. 71:169-181). Herein we describe the results of further in vitro studies investigating the effects of MAPKAP kinase-2 phosphorylation on vimentin and the effects of the phosphorylation on the filamentous structure. We show that MAPKAP kinase-2 mainly phosphorylates vimentin at Ser-38, Ser-50, Ser-55, and Ser-82, residues all located in the head domain of the protein. Surprisingly, and in stark contrast to phosphorylation by most other kinases, phosphorylation of vimentin by MAPKAP kinase-2 has no discernable effect on its assembly. It suggested that structure disassembly is not the only obligated consequence of phosphorylated vimentin as regulated by other kinases. Finally, a mutational analysis of each of the phosphorylated serine residues in vimentin suggested that no single serine site was primarily responsible for structure maintenance, implying that the retention of filamentous structure may be the result of the coordinated action of several phosphorylated serine sites. This also shed new lights on the functional task(s) of vimentin that is intermediate filament proteins might provide a phosphate reservoir to accommodate the phosphate surge without any structural changes.     

Analysis of transglutaminase protein substrates by functional proteomics

Transglutaminases are calcium-dependent enzymes that catalyze a post-translational modification of proteins through the formation of epsilon -(gamma-glutamyl)lysine bonds. Although specific roles for transglutaminases have been described, recent findings have provided evidence that dysregulation of transglutaminases may contribute to many pathological processes including celiac disease and neurodegenerative diseases. A crucial step in the elucidation of biological and pathological roles of transglutaminases requires the identification of protein substrates. A strategy based on a functional proteomic analysis was set up using two well-characterized biotinylated transglutaminase substrates as affinity probes: 5-(biotinamido)pentylamine and the synthetic biotinylated peptide TVQQEL, the amino- and acyl-donor probes, respectively. A pool of known tissue type transglutaminase protein substrates was selected in order to test the procedure. Results obtained in this paper indicate that the whole strategy can be successfully applied in order to identify transglutaminases protein substrates as well as the amino acid site sensitive toward enzyme activity.     

Phosphatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinase activation associated with hepatocyte growth factor-induced cell scattering

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration. EGF, which failed to induce cell scattering, caused a short-term increase in the phosphorylation of Erk1/Erk2 MAP kinases. On the contrary, HGF or phorbol ester stimulated the phosphorylation of MAP kinases for a long time. Experiments performed with LY294002 indicated that phosphatidylinositol 3-kinase contributed to the HGF-stimulated phosphorylation of Erk1/Erk2. This finding was confirmed by the demonstration that the MAP kinase cascade-dependent expression of a high-Mr (>300 kDa) protein pair appearing in the course of cell scattering was inhibited by LY294002 in HGF-induced cells but was not inhibited in phorbol ester-treated cells.     

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic (32)P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.     

Adenosine induces G2/M cell‐cycle arrest by inhibiting cell mitosis progression

Cellular adenosine accumulates under stress conditions. Few papers on adenosine are concerned with its function in the cell cycle. The cell cycle is the essential mechanism by which all living things reproduce and the target machinery when cells encounter stresses, so it is necessary to examine the relationship between adenosine and the cell cycle. In the present study, adenosine was found to induce G₂/M cell‐cycle arrest. Furthermore, adenosine was found to modulate the expression of some important proteins in the cell cycle, such as cyclin B and p21, and to inhibit the transition of metaphase to anaphase in mitosis.