Association of Immature Hypophosphorylated Protein Kinase Cε with an Anchoring Protein CG-NAP

Abstract

Protein kinase C (PKC) family requires phosphorylation of itself to become competent for responding to second messengers. Much attention has been focused on elucidating the role of phosphorylation in PKC activity; however, it remains unknown where this modification takes place in the cells. This study examines whether anchoring protein is involved in the regulation of PKC phosphorylation. A certain population of PKC⑀ in rat brain extracts as well as that expressed in COS7 cells was associated with an endogenous anchoring protein CG-NAP (centrosome and Golgi localized PKN-associated protein). Pulse chase experiments revealed that the associated PKC⑀ was an immature species at the hypophosphorylated state. In vitro binding studies confirmed that non-or hypophosphorylated PKC⑀ directly bound to CG-NAP via its catalytic domain, whereas sufficiently phosphorylated PKC⑀ did not. PKC⑀ mutant at a potential phosphorylation site of Thr-566 or Ser-729 to Ala, possessing almost no catalytic activity, was associated and co-localized with CG-NAP at Golgi/centrosome area. On the other hand, wild type and a phosphorylation-mimicking mutant at Thr-566 were mainly distributed in cytosol and represented second messenger-dependent catalytic activation. These results suggest that CG-NAP anchors hypophosphorylated PKC⑀ at the Golgi/centrosome area during maturation and serves as a scaffold for the phosphorylation reaction. PKC1 family is a key player in the cellular responses mediated by the second messenger diacylglycerol and the phorbol ester tumor promoters (1). Although the allosteric control is an essential feature of its action as a signal transducer, it has been revealed that an additional two types of control are also important as follows: one is phosphorylation of kinases themselves (2, 3), and the other is specific subcellular localization mediated by anchoring proteins (4, 5). The PKC family has three conserved in vivo phosphorylation sites as follows: the activation loop site and two sites located C-terminal to the kinase domain (6, 7), with the exception of the atypical PKC isozymes having a Glu residue at the most carboxyl position. Modification of these sites by combination of trans-and autophosphorylation is required to gain catalytically competent conformation in order to respond to second messengers (8 -11). It has been found that the activation loop site of PKC as well as other kinases such as PKB and p70S6K is phosphorylated by phosphoinositide-dependent protein kinase 1 (PDK1) (12-15). Because PDK1 kinase activity is constitutive (16), activation loop phosphorylation may be controlled by localization and conformation of the substrates. For instance, phosphorylation of PKB is triggered by translocation to the membrane through binding its pleckstrin homology domain with phosphatidylinositol-(3,4,5)-triphosphate, causing a conformational change that exposes the activation loop phosphorylation site (17). It is likely that the PKC family is also recruited to the particular place through binding with lipid or with anc...

A-kinase anchoring proteins and neuronal signaling mechanisms

“…Historically, the evidence to support their function has been gleaned from analysis of PKA phosphorylation events; however, AKAP-mediated compartmentalization of other signaling enzymes is equally important. For example, AKAP350/CG-NAP, AKAP220, and WAVE-1 assemble functional protein networks that contribute to Rho kinase signaling, glycogen synthase kinase 3 action, and Rac-mediated actin remodeling, respectively (Takahashi et al 2000;Soderling et al 2002;Tanji et al 2002). Another example is the AKAP79/150-mediated anchoring of PKC␤ for a role in the suppression of M currents in SCG neurons .…”

Acute Functional Tolerance to Ethanol Mediated by Protein Kinase Cɛ

“…This permits autophosphorylation at T710 and subsequent phosphorylation at S729 either by autophosphorylation (Cenni et al, 2002) or by a different kinase (Parekh et al, 1999). Phosphorylation at S729 permits full activity of PKCe when the enzyme is stimulated by lipid second messengers (Parekh et al, 1999;Takahashi et al, 2000). Thus, PKCe phosphorylation at S729 has been used as a marker for activation and posttranslational processing of the immature pool of PKCe that is not yet fully phosphorylated (Saitoh et al, 2001;Bayer et al, 2003;Zhou et al, 2003;Olive et al, 2005).…”

“…Unlike conventional PKCa, -b, or -g isozymes, which are constitutively phosphorylated, there is a pool of unphosphorylated and inactive PKCe that is stable within cells and can be recruited into cell signaling pathways by activation of phosphoinositide 3-kinase, which leads to the phosphorylation of the activation loop of PKCe at T566 by phospholipid- dependent kinase 1 (Cenni et al, 2002). Phosphorylation at this site is required for enzymatic activity (Parekh et al, 1999;Takahashi et al, 2000). This permits autophosphorylation at T710 and subsequent phosphorylation at S729 either by autophosphorylation (Cenni et al, 2002) or by a different kinase (Parekh et al, 1999).…”

“…As there are no specific substrates of PKCe that have been identified, it is not yet possible to assay PKCe activity in intact cells or tissues. PKCe, like other PKC isozymes, undergoes a series of post-translational phosphorylation events that result in a mature, phosphorylated enzyme capable of being fully activated by lipid second messengers (Newton, 2003) and these events can be assayed using phosphorylation-site-specific anti-PKCe antibodies (Parekh et al, 1999;Takahashi et al, 2000;Cenni et al, 2002). Unlike conventional PKCa, -b, or -g isozymes, which are constitutively phosphorylated, there is a pool of unphosphorylated and inactive PKCe that is stable within cells and can be recruited into cell signaling pathways by activation of phosphoinositide 3-kinase, which leads to the phosphorylation of the activation loop of PKCe at T566 by phospholipid- dependent kinase 1 (Cenni et al, 2002).…”

Intracellular Targeting of Protein Kinases and Phosphatases

“…A common scenario is the clustering of protein kinase and phosphatase activities. For example, AKAP450/CG-NAP, a large centrosomal AKAP of unknown function, has been reported to bind three kinases (protein kinase [PK] A, C, and N) and two phosphatases (PP1 and PP2A) (37)(38)(39). Likewise, the simultaneous association of PP1 and PKA with anchoring proteins such as AKAP149/D-AKAP-1 and AKAP 220 undoubtedly contributes to the bidirectional regulation of phosphorylation events at mitochondria, endoplasmic reticulum, and vesicular organelles (40,41).…”