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)...
There are 13 Dictyostelium Src homology 2 (SH2) domain proteins, almost 10-fold fewer than in mammals, and only three are functionally unassigned. One of these, LrrB, contains a novel combination of protein interaction domains: an SH2 domain and a leucine-rich repeat domain. Growth and early development appear normal in the mutant, but expression profiling reveals that three genes active at these stages are greatly underexpressed: the ttdA metallohydrolase, the abcG10 small molecule transporter, and the cinB esterase. In contrast, the multigene family encoding the lectin discoidin 1 is overexpressed in the disruptant strain. LrrB binds to 14-3-3 protein, and the level of binding is highest during growth and decreases during early development. Comparative tandem affinity purification tagging shows that LrrB also interacts, via its SH2 domain and in a tyrosine phosphorylation-dependent manner, with two novel proteins: CldA and CldB. Both of these proteins contain a Clu domain, a >200-amino acid sequence present within highly conserved eukaryotic proteins required for correct mitochondrial dispersal. A functional interaction of LrrB with CldA is supported by the fact that a cldA disruptant mutant also underexpresses ttdA, abcG10, and cinB. Significantly, CldA is itself one of the three functionally unassigned SH2 domain proteins. Thus, just as in metazoa, but on a vastly reduced numerical scale, an interacting network of SH2 domain proteins regulates specific Dictyostelium gene expression.
Sporotrichum thermophile grew well and produced plant cell-wall degrading enzymes on straw (barley and wheat) of different particle sizes and Avicel as carbon sources. Comparable activities of endoglucanase, Avicelase and cellobiase were produced on each substrate. In contrast, activities of xylanase, aryl-β-glucosidase, β-xylosidase, esterase and α-L-arabinofuranosidase were higher on straw (either wheat or barley) than on Avicel. The enzyme systems produced on barley straw of different particle sizes degraded finely milled barley straw in vitro more rapidly and to a greater extent than those produced on Avicel. In contrast, the enzyme systems produced on Avicel and very coarse barley straw hydrolysed Avicel to about the same extent while that produced on fine barley straw was slightly less effective. The main hydrolysis product in all cases was glucose. Isoelectric focusing revealed that the plant cell-wall degrading enzyme system produced by S. thermophile on barley straw was qualitatively and quantitatively superior to that produced on Avicel.
Dictyostelium is the only non-metazoan with functionally analyzed SH2 domains and studying them can give insights into their evolution and wider potential. LrrB has a novel domain configuration with leucine-rich repeat, 14-3-3 and SH2 protein–protein interaction modules. It is required for the correct expression of several specific genes in early development and here we characterize its role in later, multicellular development. During development in the light, slug formation in LrrB null (lrrB-) mutants is delayed relative to the parental strain, and the slugs are highly defective in phototaxis and thermotaxis. In the dark the mutant arrests development as an elongated mound, in a hitherto unreported process we term dark stalling. The developmental and phototaxis defects are cell autonomous and marker analysis shows that the pstO prestalk sub-region of the slug is aberrant in the lrrB- mutant. Expression profiling, by parallel micro-array and deep RNA sequence analyses, reveals many other alterations in prestalk-specific gene expression in lrrB- slugs, including reduced expression of the ecmB gene and elevated expression of ampA. During culmination ampA is ectopically expressed in the stalk, there is no expression of ampA and ecmB in the lower cup and the mutant fruiting bodies lack a basal disc. The basal disc cup derives from the pstB cells and this population is greatly reduced in the lrrB- mutant. This anatomical feature is a hallmark of mutants aberrant in signaling by DIF-1, the polyketide that induces prestalk and stalk cell differentiation. In a DIF-1 induction assay the lrrB- mutant is profoundly defective in ecmB activation but only marginally defective in ecmA induction. Thus the mutation partially uncouples these two inductive events. In early development LrrB interacts physically and functionally with CldA, another SH2 domain containing protein. However, the CldA null mutant does not phenocopy the lrrB- in its aberrant multicellular development or phototaxis defect, implying that the early and late functions of LrrB are affected in different ways. These observations, coupled with its domain structure, suggest that LrrB is an SH2 adaptor protein active in diverse developmental signaling pathways.
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