Search

Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription–quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR. [ABSTRACT FROM AUTHOR]

Highlights: [•] MAPK signalling involved in pearl millet's defence against downy mildew. [•] Differential number of kinases induced by pathogen in the two pearl millet cultivars. [•] Pathogen induced a rapid and increased activation of MAPKs in resistance cultivar. [•] Maximum activation of PgMPKs paralleled activation of defence enzymes and H2O2. [•] β-Amino butyric acid and H2O2 were found to be best activators of PgMPK1. [Copyright &y& Elsevier]

Abstract: The protozoan parasite Leishmania donovani undergoes various developmental transitions during its infectious cycle that are triggered by environmental signals encountered inside insect and vertebrate hosts. Intracellular differentiation of the pathogenic amastigote stage is induced by pH and temperature shifts that affect protein kinase activities and downstream protein phosphorylation. Identification of parasite proteins with phosphotransferase activity during intracellular infection may reveal new targets for pharmacological intervention. Here we describe an improved protocol to trace this activity in L. donovani extracts at high resolution combining in-gel kinase assay and two-dimensional gel electrophoresis. This 2D procedure allowed us to identify proteins that are associated with amastigote ATP-binding, ATPase, and phosphotransferase activities. The 2D in-gel kinase assay, in combination with recombinant phospho-protein substrates previously identified by phospho-proteomics analyses, provides a novel tool to establish specific protein kinase–substrate relationships thus improving our understanding of Leishmania signal transduction with relevance for future drug development. [Copyright &y& Elsevier]

Mitogen-activated protein kinase (MAPK) plays an important role in mediating the intracellular transmission and amplification of extracellular stimuli. We examined whether MAPK is involved in the signaling process during the early step of nodule formation. A genistein induced culture filtrate (GCF) of Bradyrhizobium japonicum was prepared for inducing an early response by soybean root hairs via Nod factor. Upon treatment, several types of deformations were seen, demonstrating that GCF contains active Nod factor molecules. In-gel kinase assays showed that treating soybean roots with GCF induced the rapid activation of two protein kinases (molecular masses of 47 kD and 44 kD), which phosphorylate myelin basic protein (MBP). To identify the activated kinase, we prepared an antibody against GMK1 (Glycine max MAP kinase 1), based on information from SIMK (an alfalfa MAP kinase) and a soybean EST database. An immunocomplex kinase assay with the GMK1-specific antibody revealed that the 47-kD kinase in GCF-treated seedlings is indeed GMK1. Consistent with many other MAP kinases, GMK1 is likely to be under post-translational regulation. Considering these results and previous reports from soybean, GMK1 seems to be a signaling mediator with a broad range of stimuli, including a fungal elicitor, wounding, and the symbiotic interaction between soybean and B. japonicum. [ABSTRACT FROM AUTHOR]

The C-terminus of latent membrane protein 1 (LMP1) can be phosphorylated in vivo. However, the protein kinase responsible for LMP1 phosphorylation has not yet been identified. In this study, GST fusion proteins containing the C-terminus of LMP1 were generated and used as substrates to survey the kinases that phosphorylate LMP1. Among several purified protein kinases tested, only protein kinase CK2 (CK2) could specifically phosphorylate LMP1. Using the in-gel kinase assay in the absence and presence of a selective CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole, CK2 was determined to be the major kinase to phosphorylate LMP1 in lymphoma and epithelial cell lines. This is the first study to show that CK2 is a potent kinase to phosphorylate LMP1 in vitro. [Copyright &y& Elsevier]

Plant physiology, in particular, is governed by a repertoire of endogenous as well as environmental cues. Auxin and cytokinin constitute an indispensable phytohormonal system required for plant growth and development. Another pivotal aspect of plant physiological process that thoroughly affects various plant growth and developmental attributes is the signaling network, majorly comprising the canonical mitogen activated protein kinase (MAPK) cascade. Striking a fine balance between the phytohormonal and signaling components could be adopted as an intricate strategy by plants to counteract various stresses in question. Thus, a brief understanding of this multifaceted complex could be of use for delineating numerous plant physiological and developmental phenomena. Thus, the present section discusses the various MAPK related assays in context to auxin and cytokinin crosstalk. Briefly, this chapter outlines the discrete MAPK methods to better understand the fundamentals of MAPK signaling network in auxin and cytokinin treated rice seedlings. Further, various phenotypic, genomic as well as proteomic protocols are discussed for a better understanding of MAPK networks in the backdrop of auxin and cytokinin interplay.

Calcium-dependent protein kinases undergo a rapid biochemical activation in response to an intracellular Ca increase induced by the PRR-dependent perception of a pathogen-related stimulus. Based on SDS gel resolution, the in-gel kinase assay allows the analysis of multiple in vivo protein samples in parallel, combining the advantage of protein separation according to molecular mass with the activity read-out of a protein kinase assay. It thus enables to follow the transient CDPK activation and inactivation in response to in vivo elicitation with a time-wise resolution. In addition, changes of CDPK phosphorylation activity often correlate with slight shifts in the enzyme's apparent molecular mass, indicating posttranslational modifications and a conformational change of the active enzyme compared to its inactive resting form. These band shifts can be detected by a simple immunoblotting to monitor CDPK activation.