No abstract

Two references cited in the text were omitted from the Literature Cited: Reddy, S.; Aggarwal, B. B. Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase. FEBS Lett. 1994, 341, 19-22. Yamamoto, H.; Hanada, K.; Kawasaki, K.; Nishijima, M. Inhibitory effect of curcumin on mammalian phos-pholipase D activity.

SummaryMutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome (RTS). A subset of RTS patients is predisposed to cancer and is sensitive to DNA damaging agents. The enhanced sensitivity of cells from RTS patients correlates with the accumulation of transcriptionally active nuclear p53. We found that in untreated normal human cells these two nuclear proteins, p53 and RECQL4, instead colocalize in the mitochondrial nucleoids. RECQL4 accumulates in mitochondria in all phases of the cell cycle except S phase and physically interacts with p53 only in the absence of DNA damage. p53-RECQL4 binding leads to the masking of the nuclear localization signal of p53. The N-terminal 84 amino acids of RECQL4 contain a mitochondrial localization signal, which causes the localization of RECQL4-p53 complex to the mitochondria. RECQL4-p53 interaction is disrupted after stress, allowing p53 translocation to the nucleus. In untreated normal cells RECQL4 optimizes de novo replication of mtDNA, which is consequently decreased in fibroblasts from RTS patients. Wild-type RECQL4-complemented RTS cells show relocalization of both RECQL4 and p53 to the mitochondria, loss of p53 activation, restoration of de novo mtDNA replication and resistance to different types of DNA damage. In cells expressing D84 RECQL4, which cannot translocate to mitochondria, all the above functions are compromised. The recruitment of p53 to the sites of de novo mtDNA replication is also regulated by RECQL4. Thus these findings elucidate the mechanism by which p53 is regulated by RECQL4 in unstressed normal cells and also delineates the mitochondrial functions of the helicase.

RecQL1 DNA helicase of the human RecQ helicase family participates in DNA repair and recombination pathways during cell-cycle replication. When we examined the effect of RecQL1 suppression on cell growth, we found that RecQL1 silencing by small interference RNA efficiently prevented proliferation of a wide range of cancer cells by inducing mitotic catastrophe and mitotic cell death. In contrast, such mitotic cell death was not seen in the growing normal fibroblasts used as controls, even if RecQL1 expression was fully downregulated. Our results support the hypothesis that endogenous DNA damage that occurs during DNA replication and remains unrepaired in cancer cells due to RecQL1 silencing induces cancer cell-specific mitotic catastrophe through a less-strict checkpoint in cancer cells than in normal cells. We speculate that normal cells are exempt from such mitotic cell death, despite slow growth, because cell-cycle progression is controlled strictly by a strong checkpoint system that detects DNA damage and arrests progression of the cell cycle until DNA damage is repaired completely. These results suggest that RecQL1 helicase is an excellent molecular target for cancer chemotherapy. (Cancer Sci 2008; 99: 71-80)

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rhokinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasindependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.