We resolved from spinach (Spinacia oleracea) leaf extracts four Ca 2؉ -independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutarylcoenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.Recent studies have defined a subfamily of plant protein kinases that are related to mammalian AMPK and the SNF1 protein kinase from the yeast Saccharomyces cerevisiae (for review, see Hardie and Carling, 1997; Halford and Hardie, 1998; Hardie et al., 1998). Mammalian AMPK switches off ATP-consuming anabolic pathways and switches on ATPproducing catabolic pathways by phosphorylating key regulatory enzymes such as HMG-CoA reductase (Corton et al., 1995). AMPK is activated by increased AMP and decreased ATP via a complex mechanism involving allosteric regulation (Corton et al., 1995), promotion of phosphorylation by an upstream protein kinase (AMPKK) (Hawley et al., 1995), and inhibition of dephosphorylation (Davies et al., 1995). Since AMP is elevated under conditions in which ATP is depleted because of the action of adenylate kinase, the kinase cascade is activated in a sensitive manner in response to cellular stresses that cause ATP depletion. We propose that AMPK acts as a "fuel gauge," protecting cells against the effects of environmental or nutritional stresses that deplete ATP (Hardie and Carling, 1997; Hardie et al., 1998).A 1992 study (MacKintosh et al., 1992) reported that extracts of several plant species contained protein kinase(s)...

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identi®ed orthologous rodent cDNAs that corresponded to new, unidenti®ed 5' splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at noncanonical sites within the body of the 3' terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3' UTR. These events constitute a novel coding arrangement and represent the ®rst report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either dier minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3' UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identi®ed Ov/Br septin isoforms by RT ± PCR con®rms a complex transcriptional pattern, with several isoforms showing tissue-speci®c distribution. To date, none of the other human septins have demonstrated such transcriptional complexity. Oncogene (2001) 20, 5930 ± 5939.

The septin family of genes has been implicated in a variety of cellular processes including cytokinesis, membrane transport and fusion, exocytosis, and apoptosis. One member of the septin family maps to chromosome 17q25.3, a region commonly deleted in sporadic ovarian and breast tumours, and has also been identified as a fusion partner of MLL in acute myeloid leukaemias. The present study demonstrates that the pattern of expression of multiple splice variants of this septin gene is altered in ovarian tumours and cell lines. In particular, expression of the zeta transcript is detectable in the majority of tumours and cell lines, but not in a range of non-malignant adult and fetal tissues. Zeta expression is accompanied by loss of the ubiquitous beta transcript. Somatic mutations of the gene were not detected in ovarian tumours, but it was demonstrated that beta expression in tumour cell lines can be reactivated by 5-azacytidine treatment, suggesting a role for methylation in the control of expression of this gene.

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