A two-tiered kinase cascade is a potentially important therapeutic target for hypertension and neurological diseases of hyperexcitability.

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser ⁶⁵ )—which lies within its ubiquitin‐like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser ⁶⁵ . How Ser ⁶⁵ ‐phosphorylated ubiquitin (ubiquitin ^{Phospho‐Ser65} ) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin ^{Phospho‐Ser65} binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser ⁶⁵ by PINK1 in vitro . Analysis of the Parkin structure, corroborated by site‐directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin ^{Phospho‐Ser65} , thereby promoting Parkin Ser ⁶⁵ phosphorylation and activation of its E3 ligase activity in vitro . Mutation of His302 markedly inhibits Parkin Ser ⁶⁵ phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin ^{Phospho‐Ser65} to Parkin disrupts the interaction between the Ubl domain and C‐terminal region, thereby increasing the accessibility of Parkin Ser ⁶⁵ . Finally, purified Parkin maximally phosphorylated at Ser ⁶⁵ in vitro cannot be further activated by the addition of ubiquitin ^{Phospho‐Ser65} . Our results thus suggest that a major role of ubiquitin ^{Phospho‐Ser65} is to promote PINK1‐mediated phosphorylation of Parkin at Ser ⁶⁵ , leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho‐Ser ⁶⁵ ‐binding pocket on the surface of Parkin that is critical for the ubiquitin ^{Phospho‐Ser65} interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin ^{Phospho‐Ser65} , which could aid in the development of Parkin activators that mimic the effect of ubiquitin ^{Phospho‐Ser65} .

Precise homoeostasis of the intracellular concentration of Cl⁻ is achieved via the co-ordinated activities of the Cl⁻ influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na⁺–K⁺ ion co-transporters), also promote inhibition of the KCCs (K⁺–Cl⁻ co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr¹⁰⁴⁸)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of ⁸⁶Rb⁺ uptake that was not markedly stimulated further by hypotonic high K⁺ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr⁵ in KCC1/KCC3 and Thr⁶ in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser⁹⁶). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl⁻ influx, we propose that the targeting of WNK–SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl⁻ extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.

Immature neurons need WNK1-dependent phosphorylation of KCC2 to prevent a premature switch in the Cl ⁻ gradient and the effect of GABA.