Dual Role of Pseudosubstrate in the Coordinated Regulation of Protein Kinase C by Phosphorylation and Diacylglycerol

Abstract

The activity of protein kinase C is reversibly regulated by an autoinhibitory pseudosubstrate, which blocks the active site of the enzyme in the absence of activators. However, before it can be allosterically regulated, protein kinase C must first be processed by three ordered phosphorylations, the first of which is modification of the activation loop catalyzed by the phosphoinositide-dependent kinase-1 (PDK-1). Here we use limited proteolysis to show that 1) newly synthesized protein kinase C adopts a conformation in which its pseudosubstrate sequence is removed from the active site, and 2) this exposure is essential to allow PDK-1 to phosphorylate the enzyme. Precursor (unphosphorylated) protein kinase C ␤II obtained by 1) in vitro transcription and translation, 2) expression of a phosphorylation-deficient mutant (T500V), or 3) in vivo labeling with a pulse of [ 35 S]cysteine/methionine is cleaved at the amino-terminal pseudosubstrate by the endoproteinase Arg-C. In marked contrast to mature (phosphorylated) enzyme, proteolysis occurs in the absence of lipid activators, revealing that precursor protein kinase C has its pseudosubstrate sequence removed constitutively. Additionally, we show that PDK-1 is unable to phosphorylate protein kinase C when the active site is sterically blocked by a peptide substrate. Neither can mature enzyme be dephosphorylated when the active site is blocked by binding either the pseudosubstrate sequence or a heterologous substrate. Thus, the accessibility of the activation loop to both phosphorylation and dephosphorylation requires an exposed pseudosubstrate. In summary, newly synthesized protein kinase C adopts a conformation in which its pseudosubstrate sequence is removed from the active site, rendering the activation loop accessible to phosphorylation by PDK-1. Phosphorylation serves as a conformational switch to position the pseudosubstrate so that it blocks the active site, a conformation that is maintained until stimulus-dependent membrane binding releases it, thus activating the enzyme.The protein kinase Cs comprise a family of enzymes that are regulated by two distinct mechanisms: phosphorylation and membrane targeting (reviewed in Ref. 1). In the first regulatory mechanism, protein kinase C isozymes are rendered catalytically competent by a series of ordered phosphorylation events, the first of which has recently been shown to be catalyzed by PDK-1 1 (2-4). PDK-1 phosphorylates a segment near the entrance to the active site referred to as the activation loop; the phosphorylated position corresponds to Thr 500 in the conventional protein kinase C ␤II (5). This phosphorylation serves to correctly position residues for catalysis and triggers the autophosphorylation of two conserved positions at the carboxyl terminus, a turn motif (Thr 641 in protein kinase C ␤II), and a hydrophobic motif (Ser 660 in protein kinase C ␤II) (6). The phosphorylated "mature" species localizes to the cytosol where it is maintained in an inactive conformation by an autoinhibitory pseudosubstrate seq...

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…This pseudosubstrate (PS) sequence binds to the substrate-binding cavity in the C-terminus and blocks catalytic activity. Removal of this pseudosubstrate sequence from the kinase domain occurs when lipid second messengers bind to the regulatory domain, resulting in substrate binding and phosphorylation [5].…”

The Phosphoinositide-dependent Kinase, PDK-1, Phosphorylates Conventional Protein Kinase C Isozymes by a Mechanism That Is Independent of Phosphoinositide 3-Kinase

“…Step in the Maturation of Protein Kinase C-Mutation of the activation loop phosphorylation sites to non-phosphorylatable, neutral residues results in expression of PKC that is not phosphorylated and cannot be activated (20,24,25). To determine whether phosphorylation by PDK-1 is a required first step in the maturation of PKC, or whether such constructs are processed by phosphorylation but then become rapidly dephosphorylated, we examined the processing of activation loop mutants by pulse-chase analysis.…”

“…Unless otherwise indicated, cells were maintained in DMEM containing 10% fetal bovine serum. Cells were transfected with one of the following expression constructs: bovine PKC ␣ (gift of Yusuf Hannun, Medical University of South Carolina), rat PKC ␤II, mutant constructs of rat PKC ␤II (T497A/ T498A/T500A (referred to as T500AAA), T641E, S660A, or S660E (20,22,23)), amino-terminal Myc-tagged rat PKC (gift of Alex Toker, Harvard Medical School), or murine Akt ␣ with an amino-terminal HA tag (gift of Alex Toker, Harvard Medical School) using the Superfect Transfection reagent (Qiagen). Untransfected COS-7 cells were used to study the endogenous PKC ␣.…”

“…Deletion of the PH domain or forced recruitment of Akt to membranes by addition of other membrane-targeting modules results in constitutive phosphorylation of Akt (17)(18)(19). The conformation of PKC is also critical for phosphorylation by PDK-1; the pseudosubstrate must be removed from the active site in order for PDK-1 to access the activation loop (20). However, it is unclear whether the intrinsic activity of PDK-1 is regulated in vivo.…”

The Human Biliverdin Reductase-based Peptide Fragments and Biliverdin Regulate Protein Kinase Cδ Activity

“…Stimulation of cells with anionic lipid second messengers/cofactors, such as phorbol 12-myristate 13-acetate (PMA) or diacylglycerol, causes conformational changes in PKCs that result in exposure of the activation loop and release of the auto-inhibitory pseudosubstrate sequence in the N-terminal regulatory domain of the protein from the active site (1,2). The pseudosubstrate domain, placed between the C2-like and C1 regions, maintains the kinase in an inactive conformation by interacting with the substrate recognition site in the catalytic domain, as is the case for all PKCs (15). The two zinc finger motifs in the N-terminal regulatory C1 domain of PKCs are the recognition motifs for the second messengers (16); hBVR is also a Zn 2ϩ metalloprotein (17).…”