We have analysed phosphorylation of the synthetic peptide AMARAASAAALARRR, and 23 variants, by mammalian, higher plant and yeast members of the SNFI protein kinase subfamily (AMP-activated protein kinase (AMPK), HMG-CoA reductase kinase (HRK-A), and SNF1 itself), and by mammalian caimodulin-dependent protein kinase I (CaMKI). These four kinases recognize motifs which are very similar, although distinguishable. Our studies define the following recognition motifs: ~MPK: qr~K

The catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase isoforin 1 (HMGRI cd) from Aruhidopsis thulium has been expressed in Escherichiu coli in a catalytically active form and purified. The high efficiency of the bacterial expression system together with the simplicity of the purification procedure used in this study resulted in the attainment of large quantities of pure enzyme (about 5 mg/l culture) with a final specific activity of up to 17 U/mg. This specific activity is higher than that reported to date for any 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) purified from a plant source.HMGRlcd activity was completely blocked by the HMGR inhibitor mevinolin (IC5,, = 12.5 nM). No significant differences were observed between the K,,, values of HMGRlcd for NADPH (71 2 7 pM) and (S)-.?-hydroxy-3-methylglutaryl-CoA (8.3 -t 1 ..5 pM) and those of pure HMGR preparations obtained from different plant sources. The purified HMGRl cd was reversibly inactivated by phosphorylation at a single site by Bmssica olrmcru HMGR kinase A, which is functionally related to the mammalian AMP-activated protein kinase. The site of phosphorylation is Ser.577 in the complete sequence of A. tlzulinntr HMGRI. The results in this paper represent the first evidence that a higher plant HMGR is regulated by direct phosphorylation, at least in a cell-free system. Our results also reinforce the view that the AMPactivated protein kinase/SNF1 family is an ancient and highly conserved protein kinase system.

We recently reported the existence of a protein kinase cascade in higher plants, of which the central component is a 3-hydroxy-3-methylglutaryl(HMG-)-CoA reductase kinase functionally related to mammalian AMP-activated protein kinase [MacKintosh, R. W., Davies, S. p., Clarke P. R., Weekes, J., Gillespie, S. G., Gibb Using a series of variants of the synthetic peptide substrate, the substrate specificities of the two forms are similar but not identical. Form B does not appear to be a proteolytic fragment of form A, and we therefore propose that it represents a closely related member of the same protein kinase sub-family.It is now clear that reversible protein phosphorylation is the major mechanism for the regulation of protein function in animal cells, its functions being particularly to mediate the response to extracellular signals such as hormones and cytokines, and to control events which occur discontinuously in the cell cycle such as DNA replication and mitosis. A third function which is just emerging may be to mediate the response of cells to environmental stresses such as heat shock or hypoxia [I]. At least in the case of cell-cycle control, the protein kinases involved appear to be highly conserved between plants and animals [3, 41. Plant cells also respond to a variety of external stimuli such as hormones, light and environmental stress. The systems which mediate these responses inside the plant cell remain largely undefined, although by analogy with animal systems it appears that protein kinases and phosphatases will play an important role. phosphoenolpyruvate carboxylase [8,9]. A number of higher plant protein kinases [lo-131 and protein phosphatases [14] have also been defined by cDNA cloning.Recently we obtained the first direct biochemical evidence for a protein kinase cascade in higher plants [15]. The protein kinase which is the central component of this cascade appears by functional criteria to be a homologue of the AMPactivated protein kinase, an activity which in mammalian cells is a component of the response to cellular stress [l] such as heat shock and hypoxia (unpublished results). Both the animal and plant kinases inactivate mammalian acetylCoA carboxylase and 3-hydroxy-3-methylglutaryl(HMG)-CoA reductase, apparently by phosphorylation at the same sites. The system also appears to be highly conserved in that, like the mammalian kinase, the plant kinase is inactivated by mammalian protein phosphatases and reactivated by mammalian kinase kinase. The one difference found to date is that the plant protein kinase is not activated by AMP. Since the plant lunase inactivates HMG-CoA reductase from potato tubers, and the phosphorylation site defined on mammalian HMG-CoA reductase is conserved in a number of plant HMG-CoA reductases, we have provisionally named the plant kinase HMG-CoA reductase kinase [15].In this study we report the further purification and characterization of the HMG-CoA reductase kinase protein from cauliflower inflorescences. During the course of this work we identified a seco...

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A review of dysembryoplastic neuroepithelial tumors (DNTs) in 14 patients over a 12-year period revealed four patients re-operated because of changes on magnetic resonance imaging (MRI) suggesting tumor recurrence or progression. In three of these, the histological features were identical to the initial DNT. In the fourth patient, persistent DNT was surrounded by WHO grade 2 oligoastrocytoma. In one of the other 10 patients, WHO grade 2 oligodendroglioma was present in white matter deep to and completely separate from a cortically based DNT. Fluorescence in situ hybridization showed codeletion of 1p and 19q in both the DNT and oligodendroglioma and oligoastrocytoma components. Deletions were not identified in any other tumor. Our findings corroborate other studies that 1p and 19q deletions are uncommon in DNT. These two unusual tumors also raise the possibility that rare DNTs may evolve into oligodendroglioma or oligoastrocytoma. DNTs with this altered biology can be identified by 1p and 19q deletion analysis.