Regulation of protein kinase Mzeta synthesis by multiple kinases in long-term potentiation

Abstract

The persistent activity of protein kinase Mzeta (PKMzeta) maintains synaptic long-term potentiation (LTP) and spatial memory, but the interactions between PKMzeta and the other protein kinases implicated in synaptic plasticity are unknown. During LTP, PKMzeta is rapidly synthesized from a PKMzeta mRNA that encodes a protein kinase Czeta (PKCzeta) catalytic domain without a regulatory domain; thus, second messengers that activate full-length PKC isoforms are not required to stimulate PKMzeta. Like other PKCs, however, PKMzeta must be phosphorylated on its activation loop by phosphoinositide-dependent protein kinase-1 (PDK1) for optimal catalytic activity. Thus, two sequential steps are required for the persistent increased PKMzeta activity that maintains LTP: de novo synthesis of PKMzeta and phosphorylation of its activation loop. Here, using a panel of antisera to phosphorylated and nonphosphorylated sites on PKMzeta, we show that PI3-kinase (phosphoinositide 3-kinase), CaMKII (Ca2+/calmodulin-dependent protein kinase II), MAPK (mitogen-activated protein kinase), PKA (protein kinase A), mTOR (mammalian target of rapamycin), all important for LTP induction, as well as preexisting PKMzeta, regulate the new synthesis of PKMzeta during LTP. In contrast, PDK1 forms a complex with PKMzeta and maintains maximal phosphorylation of its activation loop. Thus, the two steps of PKMzeta formation serve separate functions in LTP: the initial regulated synthesis of PKMzeta is the site of convergence and integration for multiple kinases of induction, whereas the constitutive phosphorylation of PKMzeta by PDK1 initiates the persistent autonomous activity that sustains maintenance.

Figure 1.

Synthesis, not phosphorylation of PKMζ is the common target of multiple protein kinases in LTP induction. A, Illustration of the phosphorylation sites and C-terminal (C-term) epitope used for antisera production. B, Specificity of the pT410 antiserum. Left, pT410 antiserum recognizes E. coli-expressed PKMζ after phosphorylation by PDK1; C-terminal antiserum recognizes both phosphorylated and nonphosphorylated PKMζ. Right, Exposure of hippocampal PKMζ to calf intestinal phosphatase eliminates pT410 immunostaining. C, Representative experiment showing PKMζ immunostaining with all three antisera increases after LTP and the blockade of the increases by application of the PI3-kinase inhibitor wortmannin. D, PKMζ immunostaining with all three antisera increases in parallel 1 h after tetanization in the carrier DMSO (0.01%). Asterisks denote p < 0.05. Inhibitors of PI3-kinase, CaMKII, MAPK, PKA, mTOR, PKC, and PKMζ (in 0.01% DMSO) block the synthesis of PKMζ (p > 0.5 between LTP and control slices for each inhibitor; n = 4), but do not affect the relative amounts of phospho-PKMζ and total PKMζ. Insets, Left, Ratios of pT410/C-terminal and pT560/C-terminal immunostaining show no change in the proportion of phosphorylated PKMζ after LTP and blockade of LTP by wortmannin. Middle, Representative fEPSPs for time points shown at the right (DMSO, 1, 2; wortmannin, 3, 4). Right, Time courses of experiments showing LTP in DMSO and blockade of LTP by kinase inhibitors (p > 0.5 between baseline responses and responses 1 h after tetanization for each inhibitor; n = 8).

Figure 2.

Constitutive phosphorylation of PKMζ by PDK1. A, PKMζ and PDK1 form a complex in hippocampal tissue. Left, Immunoprecipitation of PKMζ from hippocampus coprecipitates PDK1; right, immunoprecipitation of PDK1 from hippocampus coprecipitates PKMζ, as detected by all three antisera. B, Hippocampal PKMζ is maximally phosphorylated on its activation loop. Left, Representative experiment showing increasing amounts of PDK1 saturate phosphorylation of PKMζ at levels that are equivalent to the phosphorylation state of endogenous hippocampal PKMζ (shown at a lower concentration). Right, Mean ± SEM of four PDK1 phosphorylation experiments (the SEM is smaller than the symbols for each data point; pT410 immunostaining at 15 PDK1 U is set at 100%; C-terminal (C-term) immunostaining of hippocampal PKMζ is set at 100%; hippocampal PKMζ, n = 7). C, Left, PKMζ immunostaining 1 h after tetanization shows all three antisera increase in parallel, relative to nontetanized control slices. Right, PKCι/λ immunostaining 1 h after tetanization shows increases in activation loop (pT403) phosphorylation relative to total PKCι/λ in the tetanized slices, as well as increases in immunostaining for all antisera relative to nontetanized slices. Inset, Increase in the proportion of activation loop phosphorylated PKCι/λ, but not PKMζ, after LTP. Asterisks and hash mark denote p < 0.05. D, Illustration of the biochemical pathways of LTP induction and maintenance. In induction, postsynaptic NMDAR activation, critical for PKMζ synthesis (Sacktor et al., 1993; Osten et al., 1996), leads to increases in Ca²⁺, which stimulates multiple kinases that are critical for PKMζ synthesis from PKMζ mRNA. The newly synthesized, nonphosphorylated PKMζ binds to the constitutively active PDK1 and is maximally phosphorylated on T410 to generate the autonomous activity maintaining LTP. PKMζ may form a positive feedback loop to sustain increases in its synthesis during maintenance (dashed line).