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Chaperone-mediated folding and assembly of myosin in striated muscle

Chaperone-mediated folding and assembly of myosin in striated muscle. Srikakulam, Rajani; Winkelmann, Donald A. (Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA). Journal of Cell Science, 117(4), 641-652 (English) 2004 Company of Biologists Ltd. CODEN: JNCSAI. ISSN: 0021-9533. DOCUMENT TYPE: Journal CA Section: 13 (Mammalian Biochemistry) De novo folding and assembly of striated muscle myosin was analyzed by expressing a GFP-tagged embryonic myosin heavy chain (GFP-myosin) in post-mitotic C2C12 myocytes using replication defective adenoviruses. In the early stages of muscle differentiation, the GFP-myosin accumulates in bright globular foci and short filamentous structures that are later replaced by brightly fluorescent myofibrils. Time-lapse microscopy shows that the intermediates are dynamic and are present in elongating and fusing myocytes and in multinucleated myotubes. Immunostaining reveals the co-localization of the mol. chaperones Hsc70 and Hsp90 with the GFP-myosin in the intermediates, but not in the mature myofibrils. Uninfected cells have similar intermediates suggesting a common pathway for myosin maturation. Two conformation-sensitive antibodies that bind the unfolded motor domain and the coiled-coil conformation of the rod demonstrate that in the intermediates, the myosin rod is folded but the motor domain is not folded. Electron microscopy reveals that the intermediates contain loose filament bundles surrounded by a protein rich matrix. Geldanamycin, a specific inhibitor of Hsp90, reversibly blocks myofibril assembly and triggers accumulation of myosin folding intermediates. We conclude that multimeric complexes of nascent myosin filaments assocd. with Hsc70 and Hsp90 are intermediates in the folding and assembly pathway of muscle myosin.

The Heat Shock Protein 90 Inhibitor Geldanamycin and the ErbB Inhibitor ZD1839 Promote Rapid PP1 Phosphatase-Dependent Inactivation of AKT in ErbB2 Overexpressing Breast Cancer Cells

The Heat Shock Protein 90 Inhibitor Geldanamycin and the ErbB Inhibitor ZD1839 Promote Rapid PP1 Phosphatase-Dependent Inactivation of AKT in ErbB2 Overexpressing Breast Cancer Cells. Xu, Wanping; Yuan, Xitong; Jung, Yun Jin; Yang, Yongping; Basso, Andrea; Rosen, Neal; Chung, Eun Joo; Trepel, Jane; Neckers, Len ( Cell and Cancer Biology Branch, National Cancer Institute, Rockville, MD, USA). Cancer Research, 63(22), 7777-7784 (English) 2003 American Association for Cancer Research. CODEN: CNREA8. ISSN: 0008-5472. DOCUMENT TYPE: Journal CA Section: 1 (Pharmacology) AKT, a serine/threonine kinase that promotes cell survival, can be activated by overexpression of the receptor tyrosine kinase ErbB2. Conversely, down-regulation of ErbB2 inhibits AKT activation. Here, we identify PP1 as a serine/threonine phosphatase that assocs. with and dephosphorylates AKT in breast cancer cells, and we show that ErbB2 inhibits PP1-dependent dephosphorylation of AKT. Inhibition of ErbB2 by either the HSP (heat shock protein) 90 inhibitor geldanamycin or the ErbB inhibitor ZD1839 in SKBR3 cells, a human breast cancer cell line overexpressing ErbB2 protein, induces a rapid and dramatic decrease in AKT activity. Decreased AKT activity occurs many hours before the HSP90-dependent decline of AKT protein but is correlated with loss of AKT phosphorylation. Decreased AKT phosphorylation is not due to blockade of AKT activation or to preferential HSP90-mediated degrdn. of phosphorylated AKT. Instead, it is caused by increased AKT dephosphorylation. Sensitivity to a panel of phosphatase inhibitors suggests involvement of the phosphatase PP1 in this process. In vitro phosphatase assay (using PP1 immunopptd. from COS7 cells transiently transfected with the wild-type protein, as well as purified PP1) confirmed that AKT is a substrate of PP1. Furthermore, endogenous PP1 and AKT assoc. 148640-14-6 and 184475-35-2 are also in the experiment. with each other in SKBR3. However, the phosphatase is phosphorylated and its activity is suppressed (detd. by in vitro assay). In contrast, ErbB2 inhibition abrogates PP1 phosphorylation and restores its activity (measured by its ability to dephosphorylate AKT in vitro). Finally, transient overexpression of constitutively active PP1 in SKBR3 cells promotes marked dephosphorylation of endogenous AKT protein. These data indicate that ErbB2 acts to preserve the phosphorylation, and hence to prolong the activation, of AKT kinase by repressing the activity of the phosphatase PP1. ErbB2 thus functions to regulate AKT kinase by simultaneously promoting its activation while inhibiting its inactivation. .