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The protein kinase D (PKD) family is regulated through multi-site phosphorylation, including autophosphorylation. For example, PKD displays in vivo autophosphorylation on Ser-742 (and Ser-738 in vitro) in the activation loop and Ser-910 in the C-tail (hPKD1 numbering). In this paper, we describe the surprising observation that PKD also displays in vitro autocatalytic activity towards a Tyr residue in the P + 1 loop of the activation segment. We define the molecular determinants for this unusual activity and identify a Cys residue (C705 in PKD1) in the catalytic loop as of utmost importance. In cells, PKD Tyr autophosphorylation is suppressed through the association of an inhibitory factor. Our findings provide important novel insights into PKD (auto)regulation., (© 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The present study investigates the electrophysiological actions of BmK M1, an alpha-like toxin purified from the venom of the scorpion Buthus martensi Karsch, on voltage-gated Na+ channels. Using the voltage clamp technique, we assessed the BmK M1 activity on the cardiac Na+ channel (hH1) functionally expressed in Xenopus oocytes. The main actions of the toxin are a concentration-dependent slowing of the inactivation process and a hyperpolarizing shift of the steady-state inactivation. This work is the first electrophysiological characterization of BmK M1 on a cloned Na+ channel, demonstrating that this toxin belongs to the class of scorpion alpha-toxins. Our results also show that BmK M1 can be considered as a cardiotoxin.

The inhibitor-1 phosphatase but not the phosphorylase phosphatase activity of a newly discovered 250 kDa polycation-stimulated (PCSM) protein phosphatase in rabbit skeletal muscle is increased up to 10-fold by a Ca2+-dependent protease. The enzyme-directed protease effect to which the PCSH and PCSL phosphatases are insensitive was progressively lost during purification of the enzyme. This could be explained by either a slow conversion of the enzyme to an active form of the enzyme with a change in specificity, or the loss of a protease-sensitive inhibitor of the inhibitor-1 phosphatase activity, resulting in a PCS phosphatase characterized by its high inhibitor-1/phosphorylase alpha activity ratio. The Ca2+-dependent protease is completely inhibited by EGTA or leupeptin.

The deinhibitor protein, responsible for the decreased sensitivity of the ATP,Mg-dependent protein phosphatase to inhibitor-1 and the modulator protein, is inactivated by cyclic AMP-dependent protein kinase and reactivated by dephosphorylation. The specificity of this reaction was tested with the ATP,Mg-dependent phosphatase in its activated or spontaneously active form, four different forms of polycation-stimulated phosphatases (PCSH, PCSM, PCSL and PCSC) and calcineurin. Only the high -Mr polycation-stimulated protein phosphatase (PCSH), but not its catalytic subunit (PCSC), shows a high degree of specificity for the deinhibitor protein. Deinhibitor phosphatase activity of PCSH is affected neither by polycations nor by Mn ions.

The phosphorylation by casein kinase TS (II) of the modulator protein of the ATP, Mg-dependent phosphatase increases after preincubation with the PCSH1 phosphatase or with the catalytic subunit of the ATP, Mg-dependent phosphatase. Dephosphorylation by the two phosphatases combined leads to the incorporation of 2 mol phosphate per mol modulator (at Ser residues). Occupancy of the ATP, Mg-dependent phosphatase phosphorylation site(s) is a negative determinant in the phosphorylation of the modulator by kinase TS. Among the PCS phosphatases PCSH1 shows the highest activity toward the 32P-Ser residues labeled by kinase TS in untreated or previously dephosphorylated modulator, while the ATP, Mg-dependent phosphatase is totally ineffective. Protamine stimulates all phosphatase activities, so that the catalytic subunit of the ATP, Mg-dependent phosphatase becomes almost as effective as the PCSC phosphatase in dephosphorylating the kinase TS sites.