Phosphorylation Events Associated with Different States of Activation of a Hepatic Cardiolipin/Protease-activated Protein Kinase

Abstract

Cardiolipin-or protease-activated protein kinase, isolated from rat liver cytosol and originally named liver PAK-1, was found to be the natural form of protein kinase N (PKN) by comparing the sequences of 43 tryptic peptides of the purified liver enzyme and determining the corresponding liver cDNA sequence. These analyses also identified (i) Arg-546 as the major site of proteolytic activation, (ii) the protease resistance of the C-terminal extension beyond the catalytic domain, and (iii) in vivo stoichiometric phosphorylation of Thr-778 in the mature enzyme. Homology modeling of the catalytic domain indicated that phosphothreonine 778 functions as an anchoring site similar to Thr-197 in cAMP-dependent protein kinase, which stabilizes an active site compatible with preferred substrate sequences of PAK-1/PKN. Sigmoidal autophosphorylation kinetics and increased S6-(229 -239) peptide kinase activity following preincubation with ATP suggested phosphorylation-dependent activation of PAK-1/PKN. The onset of activation corresponded with phosphorylation of the regulatory domain site Ser-377 (located within a spectrin homology region), followed by Thr-504 (within a limited protein kinase C homology region), and, to a lesser extent, Thr-64 (in the RhoA-binding region). Several additional sites in the hinge region adjacent to a PEST protein degradation signal were selectively autophosphorylated following cardiolipin activation. Overall, these observations suggest that the regulation of this class of protein kinase involves complex interactions among phosphorylation-, lipid-, and other ligand-dependent activation events.The recent discovery of a phospholipid-activated protein kinase, structurally distinct from the known PKCs, 1 particularly in its regulatory domain, identified a new subclass of serine/ threonine kinase variously named the liver PAK (1-4), PKN (5, 6), and PRK (7, 8) kinases. Naturally occurring enzymes of this type were first identified in the rat liver cytosol fraction (1-4) and, more recently, in various subcellular fractions of rat testis (9). The liver enzyme was detected as a protease-activated kinase activity, hence its original name, liver protease-activated kinase-1 (PAK-1) (1-3). Its apparent M r (116,000) (3) and the matching amino acid sequences of catalytic domain-derived tryptic peptides suggest a close structural relationship with rat lung PKN (3, 5). The liver enzyme's narrow range of substrates and strong preference for basic amino acid residues in the vicinity of the phosphorylation site (1, 2) suggest a highly specialized catalytic activity with properties distinct from, although overlapping, those of the PKCs (2, 3, 14, 15). Other functional properties also distinguish liver PAK-1 from the PKCs, including differential lipid responsiveness and inhibitor sensitivities (2)(3)(4)14). In addition to lipid activation (10, 11), human PKN/PRK1 interacts specifically with, and is activated by, the RhoA GTP-binding protein in vitro and is activated via a RhoA-dependent signal transduction pathway in...

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton

“…Phosphopeptide mapping of rat PKN revealed at least seven autophosphorylation sites (17), five of which (Thr 64 , Ser 372 , Thr 534 , Ser 576 , and Ser 608 ) are conserved in the human homolog. To avoid the high background encountered with autophosphorylation in studies of in vitro phosphorylation, we examined the PDK1-catalyzed phosphorylation of an amino-terminal truncated mutant of PKN (AF3-PKN; ref.…”

“…However, whether this enzyme is activated by growth factorstimulated phosphorylation remains unclear. PKN is phosphorylated in intact cells (17), and its activity is inhibited by dephosphorylation (23). PKN also contains a putative phosphorylation motif in the activation loop highly homologous to that of Akt and other downstream substrates for PDK1 (Fig.…”

“…Kinase assays were carried out at 30°C for 5 min in the presence of PKN kinase buffer (final volume 30 l), 2 Ci [␥-32 P]ATP, phosphorylated or nonphosphorylated PKN, and the ribosomal S6 peptide (amino acids 229-239). This peptide previously has been shown to be a preferred substrate for PKN (17), but not for PDK1 (data not shown).…”

PRK1 Is Targeted to Endosomes by the Small GTPase, RhoB

“…These fractions were of particular interest as the PRK1 showed the presence of additional, slower migrating forms on SDS-PAGE. PRK1 has been shown to become heavily phosphorylated upon activation (11), and these slower migrating species appear to correspond to hyperphosphorylated forms of the enzyme. 3 The slowest migrating form of PRK1 was present in Fraction 10 from the gradient, which contains insoluble cytoskeletal proteins.…”

A Rho-Binding Protein Kinase C-Like Activity Is Required for the Function of Protein Kinase N in Drosophila Development

“…Activated Rho as well as Rac can also bind and activate the PKN-related PRK2 protein (Quilliam et al 1996;Vincent and Settleman 1997). PKN is also activated by unsaturated fatty acids such as arachadonic acid and by autophosphorylation Peng et al 1996). Phosphorylation on amino acid S377 is required for PKN localization to the plasma membrane and is essential for PKN to function as a Rho effector in mammalian cells (Zhu et al 2004).…”