Comparative genomic analysis of mitogen activated protein kinase gene family in grapevine

Mitogen activated protein kinases (MAPKs) are important proteins involved in the signal transduction of extracellular information to intracellular targets, and play a crucial role in the response to biotic and abiotic stresses. Although Arabidopsis MAPKs are used for identification of the putative MAPKs in higher plants, no grapevine MAPK gene nomenclature has yet been appeared in the literature. In this study, we have identified 12 members of grapevine MAPK gene (VvMPK) family via In-silico analysis of current grapevine genome database. The structural comparison of 12 VvMPKs through the analysis of chromosome locations, sequence annotation and paralogous gene pair indicated that VvMPKs have evolved by segmental duplication, rather than by tandem amplification. Although further functional analysis of VvMPKs through in vivo and in vivo experiments will be required, our study provides the basis for future research on the diverse signaling pathways medicated by MAPKs in grapevine.     

Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling

Understanding the process of wound healing will provide valuable insight for the development of new strategies to treat diseases associated with improper regeneration, such as blindness induced by corneal scarring. Heparan sulfate proteoglycans (HSPG) are not normally expressed in the corneal stroma, but their presence at sites of injury suggests their involvement in the wound healing response. Primary cultured corneal stromal fibroblasts constitutively express HSPG and represent an injured phenotype. Recently, nuclear localization of HSPG was shown to increase in corneal stromal fibroblasts plated on fibronectin (FN), an extracellular matrix protein whose appearance in the corneal stroma correlates with injury. One possible role for the nuclear localization of HSPG is to function as a shuttle for the nuclear transport of heparin-binding growth factors, such as basic fibroblast growth factor (FGF-2). Once in the nucleus, these growth factors might directly modulate cellular activities. To investigate this hypothesis, cells were treated with (125)I-labelled FGF-2 under various conditions and fractionated. Our results show that nuclear localization of FGF-2 was increased in cells plated on FN compared to those on collagen type I (CO). Interestingly, FGF-2-stimulated proliferation was increased in cells plated on FN compared to CO and this effect was absent in the presence of heparinase III. Furthermore, pre-treatment with heparinase III decreased nuclear FGF-2, and CHO cells defective in the ability to properly synthesize heparan sulfate chains showed reduced nuclear FGF-2 indicating that the heparan sulfate chains of HSPG are critical for this process. HSPG signaling, particularly through the cytoplasmic tails of syndecans, was investigated as a potential mechanism for the nuclear localization of FGF-2. Treatment with phorbol 12-myristate-13-acetate (PMA), under conditions that caused downregulation of protein kinase Calpha (PKCalpha), decreased nuclear FGF-2. Using pharmacological inhibitors of specific PKC isozymes, we elucidated a potential mode of regulation whereby PKCalpha mediates the nuclear localization of FGF-2 and PKCdelta inhibits it. Our studies suggest a novel mechanism in which FGF-2 translocates to the nucleus in response to injury.     

Synthesis of robust tunable oscillators using mitogen activated protein kinase cascades

Mitogen activated protein kinase (MAPK) cascade is evolutionally preserved in all eukaryotic cells, and regulates various cellular activities such as gene expression, mitosis, differentiation, and apoptosis. Recently, Bashor et al. have shown that Ste5 scaffold protein can be used to reshape the MAPK cascade through engineered feedback loops, and have used heuristic tuning mechanisms to synthesize the feedback. A problem of interest is to determine whether information regarding the underlying biochemical reactions can be used to synthesize robust feedback that will ensure that the resultant circuit has the desired properties. In this paper, we consider the problem of engineering feedback in MAPK cascade to synthesize an oscillator of the desired frequency. Our approach builds on the MAPK cascade model derived by Chikarmane et al. who have exploited the existence of a Hopf bifurcation point in the Markevich model of the MAPK cascade to synthesize the exciting kinase as a function of the doubly phosphorylated protein. We show how the L₁\mathcal{L}_1-control theory can be used for a robust synthesis of the oscillator and present the simulation results.     

Structural alteration of cell surface heparan sulfate through the stimulation of the signaling pathway for heparan sulfate 6-O-sulfotransferase-1 in mouse fibroblast cells

Heparan sulfate (HS) is a randomly sulfated polysaccharide that is present on the cell surface and in the extracellular matrix. The sulfated structures of HS were synthesized by multiple HS sulfotransferases, thereby regulating various activities such as growth factor signaling, cell differentiation, and tumor metastasis. Therefore, if the sulfated structures of HS could be artificially controlled, those manipulations would help to understand the various functions depending on HS. However, little knowledge is currently available to realize the mechanisms controlling the expression of such enzymes. In this study, we found that the ratio of 6-O-sulfated disaccharides increased at 3?h after adrenaline stimulation in mouse fibroblast cells. Furthermore, adrenaline-induced up-regulation of HS 6-O-sulfotransferase-1 (6-OST-1) was controlled by Src-ERK1/2 signaling pathway. Finally, inhibiting the signaling pathways for 6-OST-1 intentionally suppressed the adrenaline-induced structural alteration of HS. These observations provide fundamental insights into the understanding of structural alterations in HS by extracellular cues.     

Quantitative proteomic analysis of cold-responsive proteins in rice

Rice is susceptible to cold stress and with a future of climatic instability we will be unable to produce enough rice to satisfy increasing demand. A thorough understanding of the molecular responses to thermal stress is imperative for engineering cultivars, which have greater resistance to low temperature stress. In this study we investigated the proteomic response of rice seedlings to 48, 72 and 96?h of cold stress at 12-14°C. The use of both label-free and iTRAQ approaches in the analysis of global protein expression enabled us to assess the complementarity of the two techniques for use in plant proteomics. The approaches yielded a similar biological response to cold stress despite a disparity in proteins identified. The label-free approach identified 236 cold-responsive proteins compared to 85 in iTRAQ results, with only 24 proteins in common. Functional analysis revealed differential expression of proteins involved in transport, photosynthesis, generation of precursor metabolites and energy; and, more specifically, histones and vitamin B biosynthetic proteins were observed to be affected by cold stress.     

Quantitative proteomic analysis of yeast DNA replication proteins

Chromatin is dynamically regulated, and proteomic analysis of its composition can provide important information about chromatin functional components. Many DNA replication proteins for example bind chromatin at specific times during the cell cycle. Proteomic investigation can also be used to characterize changes in chromatin composition in response to perturbations such as DNA damage, while useful information is obtained by testing the effects on chromatin composition of mutations in chromosome stability pathways. We have successfully used the method of stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomic analysis of normal and pathological changes to yeast chromatin. Here we describe this proteomic method for analyzing changes to Saccharomyces cerevisiae chromatin, illustrating the procedure with an analysis of the changes that occur in chromatin composition as cells progress from a G1 phase block (induced by alpha factor) into S phase (in the presence of DNA replication inhibitor hydroxyurea).     

Structural determinants of heparan sulfate interactions with Slit proteins

We have previously demonstrated that the Slit proteins, which are involved in axonal guidance and related processes, are high-affinity ligands of the heparan sulfate proteoglycan glypican-1. Glypican-Slit protein interactions have now been characterized in greater detail using two approaches. The ability of heparin oligosaccharides of defined structure (ranging in size from disaccharide to tetradeccasaccharide) to inhibit binding of a glypican-Fc fusion protein to recombinant human Slit-2 was determined using an ELISA. Surface plasmon resonance (SPR) spectroscopy, which measures the interactions in real time, was applied for quantitative modeling of heparin-Slit binding on heparin biochips. Heparin was covalently immobilized on these chips through a pre-formed albumin-heparin conjugate, and the inhibition of Slit binding by heparin, LMW heparin, and heparin-derived oligosaccharides (di-, tetra-, hexa-, and octa-) was examined utilizing solution competition SPR. These competition studies demonstrate that the smallest heparin oligosaccharide competing with heparin binding to Slit was a tetrasaccharide, and that in the ELISA maximum inhibition (approximately 60% at 2 microM concentration) was attained with a dodecasaccharide.     

Molecular profiles of mitogen activated protein kinase signaling pathways in orofacial development

BACKGROUND: Formation of the mammalian orofacial region involves multiple signaling pathways regulating sequential expression of and interaction between molecular signals during embryogenesis. The present study examined the expression patterns of members of the MAPK family in developing murine orofacial tissue. METHODS: Total RNA was extracted from developing embryonic orofacial tissue during gestational days (GDs) 12-14 and used to prepare biotinylated cDNA probes, which were then denatured and hybridized to murine MAPK signaling pathways gene arrays. RESULTS: Expression of a number of genes involved in the (ERK1/2) cascade transiently increased in the embryonic orofacial tissue over the developmental period examined. Numerous members of the SAPK/JNK cascade were constitutively expressed in the tissue. Genes known to play a role in p38 MAPK signaling exhibited constitutive expression during orofacial development. Western blot analysis demonstrated that ERK2/1, p38, and SAPK/JNK kinases are present in embryonic orofacial tissue on each of GD 12, 13, and 14. By using phospho-specific antibodies, active ERK was shown to be temporally regulated during orofacial development. Minimal amounts of active p38 and active SAPK/JNK were detected in orofacial tissue during GDs 12-14. CONCLUSIONS: Our study documents specific expression patterns of genes coding for proteins belonging to the ERK1/2, p38, and SAPK/JNK MAPK families in embryonic orofacial tissue. We also demonstrate that active, phosphorylated forms of ERK1/2 only were detected in the embryonic tissue investigated, suggesting a more central role for members of this family in embryonic orofacial development.     

Aminocyanopyridine inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

A class of inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2) was discovered. These compounds have demonstrated activity against the enzyme with IC50 values as low as 130 nM and suppress the expression of TNFalpha in U937 cells. These represent the first small molecule inhibitors of MK-2 to be reported.     

Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR.     

Effects of phosphatidylserine on p38 mitogen activated protein kinase,cyclic AMP responding element binding protein and nuclear factor-kappaB activation in resting and activated microglial cells

In the last few years, the interaction between phosphatidylserine (PS), a phospholipid that becomes permanently exposed on the external cell surface in the early phases of apoptosis, and its specific receptor (PtdSerR) has emerged as a crucial event for the engulfing of apoptotic cells and for preventing the acquisition of pro-inflammatory functions by peripheral macrophages. Recently, we demonstrated that PtdSerR is expressed in microglial cultures purified from neonatal rat brain, and that PS-liposomes, used to mimic apoptotic cells, strongly reduce the lipopolysaccharide (LPS)-induced release of inflammatory mediators. Here, we show that in resting microglia, PS-liposomes induce cyclic AMP responding element binding protein (CREB) phosphorylation but do not activate nuclear factor-kappaB (NF-kappaB) and p38 mitogen-activated protein kinase (p38), in line with the non-inflammatory consequences of the recognition and removal of apoptotic cells by macrophages. In LPS-activated microglia, PS-liposomes did not affect NF-kappaB activation but inhibited the phosphorylation of p38 and delayed that of CREB. To our knowledge, this is the first biochemical evidence of the molecular signaling evoked by PS/PtdSerR interaction possibly related to repression of pro-inflammatory activities in microglial cells.     

Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2.

Proteins that belong to the fibroblast growth factor (FGF) family regulate proliferation, migration, and differentiation of many cell types. Several FGFs, including the prototype factors FGF-1 and FGF-2, depend on interactions with heparan sulfate (HS) proteoglycans for activity. We have assessed tissue-derived HS fragments for binding to FGF-1 and FGF-2 to identify the authentic saccharide motifs required for interactions. Sequence information on a range of N-sulfated HS octasaccharides spanning from low to high affinity for FGF-1 was obtained. All octasaccharides with high affinity for FGF-1 (> or =0.5 m NaCl required for elution) contained an internal IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3))-IdoUA(2-OSO(3))-trisaccharide motif. Octasaccharides with a higher overall degree of sulfation but lacking the specific trisaccharide motif showed lower affinity for FGF-1. FGF-2 was shown to bind to a mono-O-sulfated HS 6-mer carrying a single internal IdoUA(2-OSO(3))-unit. However, a di-O-sulfated -IdoUA(2-OSO(3))-GlcNSO(3)-IdoUA(2-OSO(3))-trisaccharide sequence within a HS 8-mer gave stronger binding. These findings show that not only the number but also the positions of individual sulfate groups determine affinity of HS for FGFs. Our findings support the notion that FGF-dependent processes can be modulated in vivo by regulated expression of distinct HS sequences.     

Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway

Musculoskeletal pain is common across all populations and costly in terms of impact on the individual and, more generally, on society. In most health-care systems, the first person to see the patient with a musculoskeletal problem such as back pain is the general practitioner, and access to other professionals such as physiotherapists, chiropractors, or osteopaths is still either largely controlled by a traditional medical model of referral or left to self-referral by the patient. In this paper, we examine the arguments for the general practitioner-led model and consider the arguments, and underpinning evidence, for reconsidering who should take responsibility for the early assessment and treatment of patients with musculoskeletal problems.     

Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway

Introduction

The c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis.

Methods

Three 2''-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis.

Results

MKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1β), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01).

Conclusions

MKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.     

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts.

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.