Protein Kinase Mζ Synthesis from a Brain mRNA Encoding an Independent Protein Kinase Cζ Catalytic Domain

Abstract

Protein kinase M (PKM) is a newly described form of PKC that is necessary and sufficient for the maintenance of hippocampal long term potentiation (LTP) and the persistence of memory in Drosophila. PKM is the independent catalytic domain of the atypical PKC isoform and produces long term effects at synapses because it is persistently active, lacking autoinhibition from the regulatory domain of PKC. PKM has been thought of as a proteolytic fragment of PKC. Here we report that brain PKM is a new PKC isoform, synthesized from a PKM mRNA encoding a PKC catalytic domain without a regulatory domain. Multiple -specific antisera show that PKM is expressed in rat forebrain as the major form of in the near absence of full-length PKC. A PKC knockout mouse, in which the regulatory domain was disrupted and catalytic domain spared, still expresses brain PKM, indicating that this form of PKM is not a PKC proteolytic fragment. Furthermore, the distribution of brain PKM does not correlate with PKC mRNA but instead with an alternate RNA transcript thought incapable of producing protein. In vitro translation of this RNA, however, generates PKM of the same molecular weight as that in brain. Metabolic labeling of hippocampal slices shows increased de novo synthesis of PKM in LTP. Because PKM is a kinase synthesized in an autonomously active form and is necessary and sufficient for maintaining LTP, it serves as an example of a link coupling gene expression directly to synaptic plasticity. LTP1 is a persistent enhancement of synaptic transmission widely studied as a physiological model of memory (1). LTP can be divided into two phases: induction, which triggers the potentiation, and maintenance, which sustains it over time. Many molecules have been implicated in LTP induction, which is initiated by the activation of N-methyl-D-aspartate (NMDA) receptors and involves several protein kinases (2). In contrast, very little is known about the molecular mechanism of maintenance. Recently, however, a specific, autonomously active form of the atypical PKC isozyme (3, 4), PKM, has been found both necessary and sufficient for maintaining LTP (5-7). Overexpression of PKM also prolongs memory in Drosophila melanogaster, suggesting it is part of an evolutionarily conserved molecular mechanism for memory storage (8).The unique role of PKM in LTP maintenance is due, in part, to its unusual structural and enzymatic properties as an autonomously active kinase. PKM consists of the independent catalytic domain of a PKC isoform (5). PKC isoforms are divided into three classes: conventional, novel, and atypical (reviewed in Refs. 9 -11). Each isoform is a single polypeptide consisting of an N-terminal regulatory domain and a C-terminal catalytic domain linked by a hinge (Fig. 1A, left). The regulatory domain contains binding sites for second messengers and an autoinhibitory pseudosubstrate sequence, which interacts with and blocks the active site of the catalytic domain. Second messengers stimulate PKC by binding to the regulatory domain, translocating th...

Cellular and subcellular localization of PKMζ

“…The production and characterization of antiserum recognizing a specific sequence (TLPPFQPQITDDYGL) within the catalytic domain of PKCz and PKMz have been described previously [3]. Absence of immunostaining was confirmed in PKMz knock-out mice (data not shown).…”

“…The cerebellum, which expresses both PKCz and PKMz [3,6], shows strong label in the molecular and granular layers and variable intensity of label of Purkinje cell bodies ( figure 1f,g). As in forebrain, white matter displays relatively weak immunoreactivity ( figure 1f,g).…”

“…PKMz is generated by an internal promoter within the z gene that produces a PKMz mRNA encoding an independent z catalytic domain [3] which is transported to the dendrites of neurons [4]. PKMz mRNA and protein are expressed almost exclusively in brain, whereas the fulllength PKCz mRNA and protein are not expressed in forebrain, except in the lateral olfactory tract [3,5,6].…”

“…In addition, during the protein synthesis-dependent, late form of LTP in the hippocampus, PKMz plays a role in the initial upregulation of new protein synthesis, including increased synthesis of PKMz itself, which is crucial for the transition from early-to late-LTP [11,12]. Following the de novo synthesis of PKMz [3], the autonomous activity of the kinase maintains synaptic potentiation for hours to days [8,9,13,14]. Similarly, after learning, the persistent activity of PKMz maintains many forms of long-term memory, including spatial [9] and trace conditioning [13] in the hippocampus and elementary associations [10,[15][16][17], extinction [18] and skilled sensorimotor memories [19] in the neocortex.…”

KIBRA Co-Localizes with Protein Kinase Mζ (PKMζ) in the Mouse Hippocampus

“…PKM lacks the regulatory domain of PKC 12) and is transcribed from an alternative promoter located in intron 4 of the mouse PKC gene, Prkcz. 13,14) PKM plays important roles in the maintenance of long-term potentiation, a cellular model of synaptic plasticity, and also in memory formation in Drosophila. [15][16][17] PKM is also critical to mammalian memory storage.…”