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Damage to mitochondria can lead to the depolarization of the inner mitochondrial membrane, thereby sensitizing impaired mitochondria for selective elimination by autophagy. However, fusion of uncoupled mitochondria with polarized mitochondria can compensate for damage, reverse membrane depolarization, and obviate mitophagy. Parkin, an E3 ubiquitin ligase that is mutated in monogenic forms of Parkinson's disease, was recently found to induce selective autophagy of damaged mitochondria. Here we show that ubiquitination of mitofusins Mfn1 and Mfn2, large GTPases that mediate mitochondrial fusion, is induced by Parkin upon membrane depolarization and leads to their degradation in a proteasome- and p97-dependent manner, p97, a AAA+ ATPase, accumulates on mitochondria upon uncoupling of Parkin-expressing cells, and both p97 and proteasome activity are required for Parkin-mediated mitophagy. After mitochondrial fission upon depolarization, Parkin prevents or delays refusion of mitochondria, likely by the elimination of mitofusins. Inhibition of Drp1mediated mitochondrial fission, the proteasome, or p97 prevents Parkin-induced mitophagy. doi/ 10.1083/jcb.201007013

Phospholemman (PLM) is a major target for phosphorylation mediated by both PKA (at Ser68) and PKC (at both Ser63 and Ser68) in the heart. In intact cardiac myocytes, PLM associates with and inhibits [Na.sup.+]-[K.sup.+]-ATPase (NKA), mainly by reducing its affinity for internal [Na.sup.+]. The inhibition is relieved upon PLM phosphorylation by PKA or PKC. The aim here was to distinguish the role of the Ser63 and Set68 PLM phosphorylation sites in mediating kinase-induced modulation of NKA function. We expressed wild-type (WT) PLM and S63A, S68A, and AA (Ser63 and Ser68 to alanine double mutant) PLM mutants in HeLa cells that stably express rat NKA-[[alpha].sub.1] and we measured the effect of PKA and PKC activation on NKA-mediated intracellular [Na.sup.+] concentration decline. PLM expression (WT or mutant) significantly decreased the apparent NKA affinity for internal [Na.sup.+] and had no significant effect on the maximum pump rate ([V.sub.max]). PKA activation with forskolin (20 [micro]M) restored NKA [Na.sup.+] affinity in cells expressing WT but not AA PLM and did not affect [V.sub.max] in either case. Similarly, PKC activation with 300 nM phorbol 12,13-dibutyrate increased NKA [Na.sup.+] affinity in cells expressing WT, S63A, and S68A PLM and had no effect in cells expressing AA PLM. Neither forskolin nor phorbol 12,13-dibutyrate affected NKA function in the absence of PLM. We conclude that PLM phosphorylation at either Set63 or Ser68 is both necessary and sufficient for completely relieving the PLM-induced NKA inhibition. FXYD; apparent Na affinity; PKA; PKC doi: 10.1152/ajpcell.00027.2010.

The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the [gamma]-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase, p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdcl. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 asa novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage. doi/ 10.1083/jcb.201001160

Cardiac [Na.sup.+]-[K.sup.+]-ATPase (NKA) regulates intracellular [Na.sup.+], which in turn affects intracellular [Ca.sup.2+] and contractility via the [Na.sup.+]/[Ca.sup.2+] exchanger. Extracellular [K.sup.+] concentration ([[K.sup.+]]) is a central regulator of NKA activity. Phospholemman (PLM) has recently been recognized as a critical regulator of NKA in the heart. PLM reduces the intracellular [Na.sup.+] affinity of NKA, an effect relieved by PLM phosphorylation. Here we tested whether the NKA [[alpha].sub.1]-vs. [[alpha].sub.2]-isoforms have different external [K.sup.+] sensitivity and whether PLM and PKA activation affects the NKA affinity for [K.sup.+] in mouse cardiac myocytes. We measured the external [[K.sup.+]] dependence of the pump current generated by the ouabain resistant NKA isoform in myocytes from wild-type (WT) mice (i.e., current due to NKA-[[alpha].sub.1]) and mice in which the NKA isoforms have swapped ouabain affinities ([[alpha].sub.1] is ouabain sensitive and [[alpha].sub.2] is ouabain resistant) to assess current due to NKA-[[alpha].sub.2]. We found that NKA-[[alpha].sub.1] has a higher affinity for external [K.sup.+] than NKA-[[alpha].sub.2] [half-maximal pump activation ([K.sub.0.5]) = 1.5 [+ or -] 0.1 vs. 2.9 [+ or -] 0.3 mM]. The apparent external [K.sup.+] affinity of NKA was significantly lower in myocytes from WT vs. PLM-knockout mice ([K.sub.0.5] = 2.0 [+ or -] 0.2 vs. 1.05 [+ or -] 0.08 mM). However, PKA activation by isoproterenol (1 [micro]M) did not alter the [K.sub.0.5] of NKA for external [K.sup.+] in WT myocytes. We conclude that 1) NKA-[[alpha].sub.1] has higher affinity for [K.sup.+] than NKA-[[alpha].sub.2] in cardiac myocytes, 2) PLM decreases the apparent external [K.sup.+] affinity of NKA, and 3) phosphorylation of PLM at the cytosolic domain does not alter apparent extracellular [K.sup.+] affinity of NKA. voltage-clamp; phosphorylation