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

Abstract

Phosphorylation by the phosphoinositide-dependent kinase, PDK-1, is required for the activation of diverse members of the AGC family of protein kinases, including the protein kinase C (PKC) isozymes. Here we explore the subcellular location of the PDK-1-mediated phosphorylation of conventional PKCs, and we address whether this phosphorylation is regulated by phosphoinositide 3-kinase. Pulse-chase experiments reveal that newly synthesized endogenous PKC ␣ is primarily phosphorylated in the membrane fraction of COS-7 cells, where it is processed to a species that is phosphorylated at the activation loop and at two carboxyl-terminal positions. This "mature" species is then released into the cytosol. Deletion of the plekstrin homology domain of PDK-1 results in a 4-fold increase in the rate of processing of PKC indicating an autoinhibitory role for this domain. Autoinhibition by the plekstrin homology domain is not relieved by binding 3-phosphoinositides; PKC is phosphorylated at a similar rate in serumtreated cells and serum-starved cells treated with the phosphoinositide 3-kinase inhibitors, LY294002 and wortmannin. Under the same conditions, the PDK-1-catalyzed phosphorylation of another substrate, Akt/protein kinase B, is abolished by these inhibitors. Our data are consistent with a model in which PDK-1 phosphorylates newly synthesized PKC by a mechanism that is independent of 3-phosphoinositides.Phosphorylation at a segment near the active site, the activation loop, critically regulates the function of diverse members of the protein kinase superfamily. Mounting evidence suggests that for AGC kinase family members, a single kinase, the phosphoinositide-dependent kinase, PDK-1, 1 assumes this role (1, 2). Originally discovered as the upstream kinase for Akt/ protein kinase B (3), PDK-1 is now known to phosphorylate many other kinases, including PKC, p70S6 kinase, ribosomal S6 kinase, p21-activated kinase, PKC-related kinase, and serum glucocorticoid kinase (1, 2, 4). The ability to regulate the function of multiple protein kinases places PDK-1 at a pivotal point in cellular signaling. Thus, understanding how the cellular function of PDK-1 is regulated is a central question in signal transduction.Signaling by PKC has been intensely studied in the past 2 decades, spawned by the seminal discovery of Nishizuka and co-workers (5) that this member of the kinase superfamily transduces the myriad of signals that promote phospholipid hydrolysis. Yet the fact that all family members depend on a series of ordered phosphorylation events before they are competent to respond to lipid second messengers has only recently been appreciated (6, 7). The discovery that PKC isozymes are processed by phosphorylation at three conserved positions (8, 9) led to a search for an upstream kinase. This search culminated in the finding that PDK-1, discovered in 1997 as the upstream kinase for the close cousin of PKC, Akt/protein kinase B, is the activation loop kinase for conventional (10), novel, and atypical PKCs (11,12). Thus, PKC is regula...

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

“…This theme developed because it was demonstrated that PKCs (predominantly cPKCs) are phosphorylated at these sites shortly after synthesis [83,42] and are often highly phosphorylated at these sites in many types of cells grown in culture, even under quiescent conditions [26,66,84]. In the absence of phosphorylation at one or more of these sites, catalytic activation of these isoforms is impaired or enzyme stability is compromised.…”

Protein kinase C as a stress sensor

“…PDK-1 is able to access its binding site at the hydrophobic motif in the kinase domain and, once docked there, it phosphorylates PKCs on the PKC activation loop. The release of PDK-1 from PKC is thought to be rate-limiting [8] but the mechanism of release is unknown. Once phosphorylated on the activation loop (Thr 514 in PKCγ) PKCs auto-phosphorylate at the turn motif and the hydrophobic motif.…”

Protein kinase Cγ in colon cancer cells: Expression, Thr514 phosphorylation and sensitivity to butyrate-mediated upregulation as related to the degree of differentiation

“…In this open conformation, PDK-1 is able to access its binding site at the hydrophobic motif in the kinase domain of the PKC and, once docked there, it phosphorylates PKCs on the activation loop. The release of PDK-1 from PKC is thought to be rate limiting [12]. Once phosphorylated on the activation loop (for PKCγ at Thr 514 ), PKCs become auto-phosphorylated at two additional sites (the turn motif and the hydrophobic motif) leading to the release of an inactive conformation from the membrane to the cytoplasm.…”

“…According to the current understanding [reviewed in 8,12], the newly-synthesized conventional PKCs associate with a membrane through interactions with the C1 and/or C2 domains. In this open conformation, PDK-1 is able to access its binding site at the hydrophobic motif in the kinase domain of the PKC and, once docked there, it phosphorylates PKCs on the activation loop.…”

“…Although the exact mechanism and role of phosphorylation in PKC priming is not yet fully understood, it was suggested that a de novo synthesized conventional PKC first binds to a membrane, enabling a conformation that permits phosphoinositide-dependent protein kinase 1 (PDK-1) [12] to bind and phosphorylate a site in the activation-loop, which is Thr 514 for PKCγ [13,14]. Phosphorylation at this site leads to a conformational change enabling phosphorylations at two carboxyl-terminal sites namely, the turn motif and the hydrophobic motif, as a result of which the fully phosphorylated conventional PKC is released from the membrane, and positioned in its primed, inactive form in the cytoplasm [15][16][17].…”