Characterization of a Low Molecular Mass Autophosphorylating Protein in Cultured Sugarcane Cells and Its Identification as a Nucleoside Diphosphate Kinase

Abstract

A low molecular m a s (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum 1.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PACE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/ SDS-PACE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36°C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. l h e amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants.Signal transduction events are likely to occur in response to environmental stresses in cells, in particular, the heatshock response. Within minutes after the onset of heat-shock conditions, altered transcription, translation, and metabolism are observed (Lindquist, 1986). These changes may involve well-known signal transduction mechanisms such as alterations in phosphatidylinositol metabolism, in cellular Ca2+ concentration (Calderwood et al., 1988), and in protein phosphorylation (Schlesinger, 1988). In sugarcane (Saccharum officinarum L.) cells, the induction of the events leading to the development of thermotolerance has a temperature optimum of 36 to 38OC with a minimum induction period of about 30

Immunological Detection of Potential Signal-Transduction Proteins Expressed during Wheat Somatic Tissue Culture

“…The low-molecularmass 16-kD putative NDPK form detected in the soluble and membrane protein fractions from wheat tissues could be related to the 16-kD form previously described (Nomura et al, 1992;Finan et al, 1994) and to the 18-kD NDPK from sugarcane cells (Moisyadi et al, 1994). The wheat immature embryos revealed a reduced amount of the 16-kD membrane form compared with in vitro-cultured cells.…”

“…The enzyme from eukaryotes is a hexamer of identical subunits with a particular mononucleotide binding fold (Dumas et al, 1992). A striking homology of the amino acid sequence of spinach NDPK with both the human and Drosophila melanogaster NDPKs was established (Nomura et al, 1992;Moisyadi et al, 1994). This led to the hypothesis of a possible contribution of the NDPK protein to the development of plant tissue, including cell proliferation, through signaltransduction pathways (Wallet at al., 1990).…”

A Novel Serine/Threonine‐Specific Protein Phosphotransferase Activity of Nm23/Nucleoside‐Diphosphate Kinase

“…A possible regulatory function remains conceivable given the abundant expression of NDPK/Nm23 in many tumor tissues [39, 401. Further evidence for the existence of a Nm23 protein phosphotransferase activity in the cell is provided by several reports on NDPK from plants, which all show a high degree of sequence similarity to human NDPK 1411. It was reported that NDPK may act as a kind of active subunit of protein kinase complexes in pea [42,43] and sugar-cane [44]. The NDPK preparations apparently exhibited a weak serine phosphotransferase activity toward membrane proteins, which could be stimulated by Ca' ' and calmodulin.…”

A 16-kDa Protein Functions as a New Regulatory Protein for Hsc70 Molecular Chaperone and Is Identified as a Member of the Nm23/Nucleoside Diphosphate Kinase Family

“…Interestingly, plant NDP kinases have been proposed as a part of the signal transduction pathway during stress (92). NDP kinase from cultured sugarcane cells exhibits heat shock-stimulated autophosphorylation activity (93), and in tomato plant, its mRNA level is up-regulated in response to wounding (94). Since one of the most common signal transduction events during heat shock is protein phosphorylation, the up-regulated NDP kinase activity and protein levels may allow the plant to perceive and transduce signals related to high temperature stress (92).…”