SUMMARY Alternative splicing of the PKM2 gene produces two isoforms, M1 and M2, which are preferentially expressed in adult and embryonic tissues, respectively. The M2 isoform is reexpressed in human cancer and has nonmetabolic functions in the nucleus as a protein kinase. Here, we report that PKM2 is acetylated by p300 acetyltransferase at K433, which is unique to PKM2 and directly contacts its allosteric activator, fructose 1,6-bisphosphate (FBP). Acetylation prevents PKM2 activation by interfering with FBP binding and promotes the nuclear accumulation and protein kinase activity of PKM2. Acetylationmimetic PKM2(K433) mutant promotes cell proliferation and tumorigenesis. K433 acetylation is decreased by serum starvation and cell-cell contact, increased by cell cycle stimulation, epidermal growth factor (EGF), and oncoprotein E7, and enriched in breast cancers. Hence, K433 acetylation links cell proliferation and transformation to the switch of PKM2 from a cytoplasmic metabolite kinase to a nuclear protein kinase.

In order to determine the functional roles of the conserved sequence (NXNSSRFG) of the "switch I" loop (residues 233-240 in Dictyostelium myosin II), alanine scanning mutagenesis was performed on Dictyostelium myosin II. N233A and S237A mutant myosins did not bind a fluorescent analog of ADP, mant-deoxyADP, at the low concentration range (micromolar and had low level of ATPase activities. They were nonmotile when examined by the in vitro motility assay. Dictyostelium cells expressing these myosins showed worse phenotypes than that of myosin-null cells. In contrast to these mutant myosins, R238A myosin tightly bound mant-deoxyADP. However, the mutant had a defect in the ATP hydrolysis step and exhibited the lowest ATPase activities among the mutants examined here. The R238A myosin was nonmotile. R238C or R238H mutations, which mimic the Usher syndrome mutations, generated myosins with similar functional defects to those of the R238A mutation. Cells expressing the R238A myosin exhibited the phenotype similar to that of the myosin-null cells. N235A, S236A, F239A, and G240A myosins retained moderate levels of ATPase activities and could drive sliding of actin filaments at various speeds. Phenotypes of cells expressing them were very similar to that of the wild-type cells. Taken together, these results suggest that side chains of N233 and S237 may play essential roles in holding a nucleotide in the ATPase pocket and that R238 may play crucial roles in the ATP hydrolysis step, while those of the other residues in the switch I loop are not essential for the process.

A loop comprising residues 454 -459 of Dictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the "switch II loop" of the myosin family is DIXGFE. In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on the Dictyostelium myosin II heavy chain gene. Examination of in vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions, whereas the D454A, G457A, F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis steps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyostelium myosin motor domain.In the Dictyostelium motor domain designated as S1dC (1), a bound nucleotide is surrounded by three loops whose sequences are highly conserved among the myosin family (2): the P-loop (residues 179 -186 of Dictyostelium myosin II) and the two loops in the 50K segment (residues 233-240 and 454 -459 of Dictyostelium myosin II) (see Fig. 1A). One of the loops in the 50K segment (residues 233-240) is homologous to a loop in the switch I region of GTPases judging from the topological similarity (3) and has the consensus sequence NXNSSRFG (NNNS-SRFG in Dictyostelium myosin II). Residues in the loop are aligned along the ATPase pocket, and some of the side chains form hydrogen bonds with the bound nucleotide. The other loop in the 50K segment has the consensus sequence, DIXGFE (DISGFE in Dictyostelium myosin II) and is functionally and structurally equivalent to a loop in the switch II region of GTPases (3). In GTPases, the switch II loop connects the GTPase site and the switch II ␣-helix, which is part of the effector binding region. Information on the nucleotide state at the GTPase site is transmitted to the effector binding region partially through this switch II loop. In myosin, the switch II loop connects the ATPase pocket and a long conserved ␣-helix embedded in the lower 50K subdomain (4, 5). Recent x-ray crystallographic studies on Dictyostelium S1dC complexed with various nucleotides and nucleotide analogs revealed that the switch II loop undergoes a significant conformational change during ATP hydrolysis (Fig. 1B) like the loop in GTPases. When S1dC is complexed with MgADP/V i or MgADP/AlFx (the V i structure), the switch II loop is closer to the ATPase pocket, although the loop moves away from the ATPase pocket when S1dC is complexed with MgADP/BeFx, MgAMPPNP, MgATP␥S, 1 or MgADP (the BeFx structure) (5-7). The observed changes in the switch II loop arise from the main chain rotation at the two pivoting residues, Ile-455 and Gly-457. In the V i structure, Gly-457 and Glu-459 are close to the bound nucleotide. G...

Brown seaweed lipids from Undaria pinnatifida (Wakame), Sargassum horneri (Akamoku), and Cystoseira hakodatensis (Uganomoku) contained several bioactive compounds, namely, fucoxanthin, polyphenols, and omega-3 polyunsaturated fatty acids (PUFA). Fucoxanthin and polyphenol contents of Akamoku and Uganomoku lipids were higher than those of Wakame lipids, while Wakame lipids showed higher total omega-3 PUFA content than Akamoku and Uganomoku lipids. The levels of docosahexaenoic acid (DHA) and arachidonic acid (AA) in liver lipids of KK-A(y) mouse significantly increased by Akamoku and Uganomoku lipid feeding as compared with the control, but not by Wakame lipid feeding. Fucoxanthin has been reported to accelerate the bioconversion of omega-3 PUFA and omega-6 PUFA to DHA and AA, respectively. The higher hepatic DHA and AA level of mice fed Akamoku and Uganomoku lipids would be attributed to the higher content of fucoxanthin of Akamoku and Uganomoku lipids. The lipid hydroperoxide levels of the liver of mice fed brown seaweed lipids were significantly lower than those of control mice, even though total PUFA content was higher in the liver of mice fed brown seaweed lipids. This would be, at least in part, due to the antioxidant activity of fucoxanthin metabolites in the liver.

Preoperative rituximab effectively decreased the anti-ABO antibodies sufficiently to prevent the AMR irrespective of splenectomy. Splenectomy does not offer any immunological benefit in ABO-I LT with preoperative rituximab.