Phosphoinositide-dependent Kinase Phosphorylation of Protein Kinase C Apl II Increases during Intermediate Facilitation inAplysia

Antonio M Pepio,Ginette L Thibault,Wayne S Sossin

doi:10.1074/jbc.m202264200

Antonio M Pepio, Ginette L Thibault + Show 1 more

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https://doi.org/10.1074/jbc.m202264200

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Abstract

Phosphorylation of protein kinase Cs (PKCs) by phosphoinositide-dependent kinase I (PDK) is critical for PKC activity. In the nervous system of the marine mollusk Aplysia, there are only two major PKC isoforms, the calcium-activated PKC Apl I and the calcium-independent PKC Apl II, and both PKCs are persistently activated during intermediate memory. We monitored the PDK-dependent phosphorylation of PKC Apl I and PKC Apl II using phosphopeptide antibodies. During persistent activation of PKCs in Aplysia neurons, there is a significant increase in the amount of PDK-phosphorylated PKC Apl II in the particulate fraction but no increase in the amount of PKC Apl I phosphorylated by PDK. PDK phosphorylation of PKCs was not sensitive to inhibitors of phosphatidylinositol 3-kinase, PKC, or expression of a kinase-inactive PDK. Localization of PDK-phosphorylated PKC Apl II using immunocytochemistry revealed an enrichment of phosphorylated PKC Apl II at the plasma membrane. These data suggest that increased PDK phosphorylation of PKC Apl II is important for persistent kinase activation.

Highlights

In the marine mollusk, Aplysia californica, PKCs1 are important for both short and intermediate term changes in synaptic strength between sensory and motor neurons that accompanies behavioral sensitization [1]
We biased the peptides used for immunization to the divergent region N-terminal of the phosphoinositide-dependent kinase I (PDK) site in order to make antibodies that are specific for individual protein kinase Cs (PKCs) isoforms and that do not recognize other PDK-phosphorylated proteins (Fig. 1A)
To evaluate the specificity of the antibodies for phosphorylated PKC, we blotted bacterially expressed PKC catalytic domain as a control because there is no PDK in bacteria, and the bacterially expressed PKC will not be phosphorylated at the PDK site

Summary

Introduction

Aplysia californica, PKCs1 are important for both short and intermediate term changes in synaptic strength between sensory and motor neurons that accompanies behavioral sensitization [1]. All PKCs require phosphorylation at the activation loop site by phosphoinositide-dependent kinase 1 (PDK) for full catalytic activity [10]. After PDK-dependent phosphorylation, Ca2ϩ-activated PKCs undergo two major autophosphorylations that are required for the stability and folding of the enzyme, a process termed maturation [9]. Once these sites are phosphorylated, the PDK site can become de-phosphorylated without affecting the stability of the enzyme [12]. In contrast to Ca2ϩ-activated PKCs [13], PDK phosphorylation of PKC␦ is regulated both by PKC autophosphorylation and by PI 3-kinase activity [15]. Regulation of phosphorylation of other kinases by PDK appears to be dependent on substrate conformation and subcellular localization as opposed to activation of PDK itself (16 –18)

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