Differential effects of fatty acid and phosholipid activators on the catalytic activities of a structurally novel protein kinase from rat liver

Abstract

The lipid responsiveness of the structurally unique protein kinase, referred to as PAK-1, recently isolated from rat liver [(1994) J. Biol. Chem. 269, in press], is characterised by the high sensitivity (low micromolar) of its ribosomal $6(229-239) peptide kinase activity to both cardiolipin and the cis-unsaturated fatty acids and insensitivity to phosphatidylserine. Autophosphorylation of PAK-1 exhibited even greater sensitivity (submicromolar) to cardiolipin, but was relatively less affected by phosphatidylserine. Oleate, the most potent activator of PAK-l's peptide kinase activity was relatively ineffectual with autophosphorylation. These and other unusual characteristics, including high levels of basal catalytic activities, suggest a novel mechanism of regulation distinct from that of the protein kinase Cs

PRK1 phosphorylates MARCKS at the PKC sites: serine 152, serine 156 and serine 163

“…Consistent with the lack of a C1 domain it has become clear that while the PRKs resemble PKCs in being activated by proteolysis [5,6], they are not activated by phorbol esters. Various fatty acids and phospholipids have now been shown to activate PRKs in vitro [6][7][8][9] although the signalling pathways leading to the activation of PRK1 in vivo remain unclear.…”

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

“…Activation of PAK-1/PKN through interaction with cardiolipin, site-specific phosphorylation, or proteolytic cleavage involves suppression of the effects of the N-terminal negative regulatory domain (3,4). The unusual structural characteristics of this domain, which presumably determine function, include a striking similarity (16 -24% amino acid identity) between extended sequences within the N-terminal leucine zipper-based heptad repeat region of the regulatory domain (residues 1-330) (5,8) and the coiled-coil motifs of contractile proteins (e.g.…”

“…The lack of clear-cut specificity for acidic phospholipid and unsaturated fatty acid activators of the PAK-1/PKN/PRK1 enzymes (3, 4, 9 -11) and the apparent inaccessibility of the cytosolic enzyme to the most potent in vitro phospholipid activator, mitochondrially located cardiolipin (3,4), suggest that these lipids may not be the physiological activators. Presumably, cardiolipin mimics the action of some yet to be identified lipid second messenger or other activating ligand.…”

“…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 cultured fibroblasts (12, 13).…”

“…Phosphorylation Site Analysis-Liver 116-kDa PAK-1 (5-15 g) was autophosphorylated with 50 M [␥- 32 P]ATP (4000 dpm/pmol) in the presence and absence of cardiolipin (10 g/ml) as described previously (4). Reaction mixtures containing autophosphorylated PAK-1 were either analyzed directly by SDS-PAGE (4) or treated with SDS (to 1%) and heated at 80°C for 5 min before desalting on a Sephadex G-25 column (1 ϫ 30 cm) equilibrated in 10 mM NH 4 HCO 3 and 0.02% SDS buffer (pH 7.8).…”