Tissue-specific differences in activation of atypical protein kinase C and protein kinase B in muscle, liver, and adipocytes of insulin receptor substrate-1 knockout mice

Insulin receptor substrates (IRSs) 1 and 2 are postulated to control the activation of phosphatidylinositol 3-kinase (PI3K)-dependent signaling factors, namely, atypical protein kinase C (aPKC) and protein kinase B (PKB)/Akt, which mediate metabolic effects of insulin. However, it is uncertain whether aPKC and PKB are activated together or differentially in response to IRS-1 and IRS-2 activation in insulin-sensitive tissues. Presently, we examined insulin activation of aPKC and PKB in vastus lateralis muscle, adipocytes, and liver in wild-type and IRS-1 knockout mice, and observed striking tissue-specific differences. In muscle of IRS-1 knockout mice, the activation of both aPKC and PKB was markedly diminished. In marked contrast, only aPKC activation was diminished in adipocytes, and only PKB activation was diminished in liver. These results suggest that IRS-1 is required for: 1) activation of both aPKC and PKB in muscle; 2) aPKC, but not PKB, activation in adipocytes; and 3) PKB, but not aPKC, activation in liver. Presumably, IRS-2 or other PI3K activators account for the normal activation of aPKC in liver and PKB in adipocytes of IRS-1 knockout mice. These complexities in aPKC and PKB activation may be relevant to metabolic abnormalities seen in tissues in which IRS-1 or IRS-2 is specifically or predominantly down-regulated.     

Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.     

Cloning and expression analysis of a MAPKKK gene and a novel nodulin gene of Lotus japonicus

We isolated a cDNA encoding mitogen-activated protein kinase kinase kinase alpha, designated LjM3Kalpha, from Lotus japonicus, a model legume. The gene was expressed constitutively in roots, root nodules, and shoots. We also identified a novel nodulin gene, LjNUF, that shows specific expression in nodules. LjNUF resembles the C-terminal half of a hypothetical protein (pir//D85436), the N-terminal half of which is similar to a portion of mitogen-activated protein kinase kinase kinase gamma. Although LjNUF was predicted to be a secreted protein, its function remains to be clarified.     

The solubilization and biological activities of Aspergillus beta-(1 --> 3)-D-glucan

We have recently demonstrated that the cell wall beta-glucan of Candida albicans could be solubilized by sodium hypochlorite, followed by dimethylsulfoxide-extraction (NaClO-DMSO method). In this study, applying this method to Aspergillus spp., we prepared mycelial cell wall beta-glucan and examined its physical properties and immunotoxicological activity. The acetone-dried mycelia of Aspergillus spp. were oxidized by the NaClO-DMSO method. An analysis of (13)C NMR spectra revealed the preparations to be composed of alpha-(1 --> 3) and beta-(1 --> 3)-D-glucan. Also, the proportion of alpha-(1 --> 3) and beta-(1 --> 3)-D-glucan varied. Furthermore, a solubilized Aspergillus beta-glucan (ASBG) was prepared from OX-Asp by urea-autoclave treatment. ASBG showed limulus activity similar to Candida solubilized beta-glucan (CSBG), and there was little difference in the activity of ASBG between various Aspergillus spp. ASBG affected the production of IL-8 by human peripheral blood mononuclear cells (PBMC). ASBG should be useful for analyzing the clinical role of beta-glucan.     

Allelic characterization of the leaf-variegated mutation <Emphasis Type="Italic">var2</Emphasis> identifies the conserved amino acid residues of FtsH that are important for ATP hydrolysis and proteolysis

Arabidopsis var1 and var2 mutants exhibit leaf variegation. VAR1 and VAR2 encode similar FtsH metalloproteases (FtsH5 and FtsH2, respectively). We have previously found many variegated mutants to be allelic to var2. Each mutant was shown to express a different degree of variegation, and the formation of white sectors was enhanced in severely variegated alleles when these alleles were grown at low temperature. VAR1/FtsH5 and VAR2/FtsH2 levels were mutually affected even in the weak alleles, confirming our previous observation that the two proteins form a hetero complex. In this study, the sites of the mutations in these var2 alleles were determined. We isolated eight point mutations. Five alleles resulted in an amino acid substitution. Three of the five amino acid substitutions occurred in Walker A and B motifs of the ATP-binding site, and one occurred in the central pore motif. These mutations were considered to profoundly suppress the ATPase and protease activities. In contrast, one mutation was found in a region that contained no obvious signature motifs, but a neighboring sequence, Gly–Ala–Asp, was highly conserved among the members of the AAA protein family. Site-directed mutagenesis of the corresponding residue in E. coli FtsH indeed showed that this residue is necessary for proper ATP hydrolysis and proteolysis. Based on these results, we propose that the conserved Gly–Ala–Asp motif plays an important role in FtsH activity. Thus, characterization of the var2 alleles could help to identify the physiologically important domain of FtsH.     

Preparation of Escherichia coli cell extract for highly productive cell-free protein expression

As structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli cell extract preparation for cell-free protein synthesis, which we have developed and routinely use. The cell extract prepared according to this protocol is used for many of our cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.