How mitochondrial metabolism is altered by oncogenic tyrosine kinases to promote tumor growth is incompletely understood. Here, we show that oncogenic HER2 tyrosine kinase signaling induces phosphorylation of mitochondrial creatine kinase 1 (MtCK1) on tyrosine 153 (Y153) in an ABL-dependent manner in breast cancer cells. Y153 phosphorylation, which is commonly upregulated in HER2 breast cancers, stabilizes MtCK1 to increase the phosphocreatine energy shuttle and promote proliferation. Inhibition of the phosphocreatine energy shuttle by MtCK1 knockdown or with the creatine analog cyclocreatine decreases proliferation of trastuzumab-sensitive and -resistant HER2 cell lines in culture and in xenografts. Finally, we show that cyclocreatine in combination with the HER2 kinase inhibitor lapatinib reduces the growth of a trastuzumab-resistant HER2 patient-derived xenograft. These findings suggest that activation of the phosphocreatine energy shuttle by MtCK1 Y153 phosphorylation creates a druggable metabolic vulnerability in cancer.
Background: Streptococcus pneumoniae serotype 11D capsular polysaccharide (CPS) structure is unknown. Results: Serotype 11D PS contains two different repeating units; one has ␣GlcNAc, and the other contains ␣Glc. Conclusion: The 11D CPS is due to the bispecific glycosyltransferase WcrL. Based on codon 112, WcrL can transfer ␣Glc, ␣GlcNAc, or both. Significance: Minimal genetic changes can make bacteria produce different polysaccharides.
For many bacteria, including those important in pathogenesis, expression of a surface-localized capsular polysaccharide (CPS) can be critical for survival in host environments. In Gram-positive bacteria, CPS linkage is to either the cytoplasmic membrane or the cell wall. Despite the frequent occurrence and essentiality of these polymers, the exact nature of the cell wall linkage has not been described in any bacterial species. Using the Streptococcus pneumoniae serotype 2 CPS, which is synthesized by the widespread Wzy mechanism, we found that linkage occurs via the reducing end glucose of CPS and the β-D-N-acetylglucosamine (GlcNAc) residues of peptidoglycan (PG). Hydrofluoric acid resistance, 31 P-NMR, and 32 P labeling demonstrated the lack of phosphodiester bonds, which typically occur in PG-polysaccharide linkages. Component sugar analysis of purified CPS-PG identified only CPS and PG sugars in the appropriate ratios, suggesting the absence of an oligosaccharide linker. Time of flight mass spectrometry confirmed a direct glycosidic linkage between CPS and PG and showed that a single CPS repeat unit can be transferred to PG. The linkage was acetolysis susceptible, indicative of a 1,6 glycosidic bond between CPS and the GlcNAc C-6. The acetylation state of GlcNAc did not affect linkage. A direct glycosidic linkage to PG was also demonstrated for serotypes 8 and 31, whose reducing end sugars are glucose and galactose, respectively. These results provide the most detailed descriptions of CPS-PG linkages for any microorganism. Identification of the linkage is a first step toward identifying the linking enzyme and potential inhibitors of its activity.capsule | Wzy | Gram positive | pneumococcus | glycobiology
A newly isolated T‐cell line (CB1) derived from a T‐acute lymphoblastic leukaemia (T‐ALL) patient contained cells (40% of total) which did not express the CD45 phosphotyrosine phosphatase. The cells were sorted into CD45‐ and CD45+ populations and shown to be clonal in origin. T‐cell receptor (TCR) cross‐linking or coligation of the TCR with its CD4/CD8 co‐receptors induced tyrosine phosphorylation and calcium signals in CD45+ but not in CD45‐ cells. Unexpectedly, whole cell p56lck and p59fyn tyrosine kinase activities were not reduced in CD45‐ compared to CD45+ cells. A novel technique was therefore developed to isolated specific pools of aggregated receptors expressed at the cell surface, together with their associated tyrosine kinases. Using this technique it was shown that cell surface CD4‐p56lck kinase activity was 78% lower in CD45‐ than in CD45+ cells. Phosphorylation of TCR zeta‐ and gamma‐chains occurred in TCR immunocomplexes from CD45+ but not CD45‐ cells, despite comparable levels of p59fyn and TCR proteins. Furthermore, TCR‐associated tyrosine kinase activity towards an exogenous substrate was 84% lower in CD45‐ than in CD45+ cells. Addition of recombinant p59fyn to TCR immunocomplexes isolated from CD45‐cells restored the phosphorylation of the TCR zeta‐ and gamma‐chains. Our results demonstrate that CD45 selectively regulates the pools of p59fyn and p56lck kinases which are associated with the TCR and CD4 at the cell surface. Activation by CD45 of these receptor‐associated kinase pools correlates with the ability of the TCR and its coreceptors to couple to intracellular signalling pathways.
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