Protein kinase C

DEVELOPMENTAL BIOLOGY

Each of the three PKC genes is expressed in the Drosophila adult head but transcripts are not detected in the body. The dPKC98F gene is expressed throughout development, with a lower level of expression in embryonic flies. The dPKC98F gene 5.5 kb transcript is greatly reduced in embryos and correlates with the increased expression of two additional transcripts of 4.3 and 4.5 kb. The dPKC98F gene is the only Drosophila PKC that is transcribed during embryonic stages. dPKC53E(brain) is transcribed in all head neural tissue. In contrast inactivation no afterpotential C or dPKC53E(ey) is specifically expressed in photoreceptor cells, both in the compound eyes and ocelli (Schaeffer, 1989).

PKC in the Adult: Function in Neurons

The calcium-dependent PKC purified from honeybee (Apis mellifera) brain shows remarkable similarities to the corresponding vertebrate PKC. Interestingly, the staining of honeybee brain with a polyclonal PKC antiserum reveals a strong, distinct labeling of the antennal lobes (ALs) and the mushroom bodies (MBs). ALs and MBs are both involved in olfactory learning in the honeybee. Local cooling experiments and local injections of octopamine into the ALs indicate that the AL may establish a memory trace independent of the MB. However, the ALs primarily process chemosensory information, and PKC immunoreactivity is located predominantly in the interneurons of the ALs. Sensory neurons projecting via the antennal nerve and innervating the rind areas of the glomeruli are stained very weakly. In contrast to the AL, the MB is involved in higher integrative functions and receives input from different sensory modalities. The MB shows high levels of PKC expression. Thus, because the ALs predominantly process olfactory information, the study concentrated on this structure of the honeybee brain to examine the role of PKC in associative olfactory learning (Grunbaum, 1998 and references).

To detect changes in PKC activity induced by in vivo stimulation, a fast phosphorylation assay was applied, using MARCKS as a PKC-specific substrate. MARCKS is phosphorylated specifically by PKC, but not by PKA or CaMII kinase from honeybee brain. MARCKS also is phosphorylated by the calcium-independent constitutively active PKC activity of PKM (generated by partial proteolysis of purified PKC). In brain homogenate that is depleted of PKC, MARCKS phosphorylation is below 10% of the phosphorylation in the original homogenate containing PKC. To confirm further the specific phosphorylation of MARCKS by PKC, the PKC inhibitor peptide was included in the phosphorylation assay. This PKC inhibitor peptide selectively inhibits MARCKS phosphorylation by PKC purified from honeybee brain without affecting PKA and CaMII kinase activity. In brain homogenates, it reduces MARCKS phosphorylation to below 10% of the phosphorylation in the absence of the inhibitor peptide. These data strongly confirm that MARCKS phosphorylation of honeybee brain homogenates is mediated exclusively by PKC (Grunbaum, 1998).

To discriminate between two different forms of PKC activation, (1) transient calcium-dependent activation and (2) constitutive calcium-independent activation, PKC activity was measured in either the presence or the absence of activators for classical PKC [calcium and diacylglycerol (DAG)]. All of the stimuli used for olfactory conditioning induce comparable transient PKC activation in the AL regardless of the stimuli and the sequence of application. A similar, although prolonged, transient activation also is induced by three forward or backward pairings with intertrial intervals (ITI) of 2 min. In the absence of PKC activators (calcium and DAG) in the in vitro assay, the PKC activity is reduced to <5% of the activity measured in the presence of the activators. This suggests that in vivo stimulation during conditioning causes a transient calcium-dependent activation of PKC in the AL (Grunbaum, 1998).

Changes in PKC activity induced by olfactory conditioning were measured in the antennal lobes. Multiple conditioning trials inducing a memory different from that induced by a single conditioning trial specifically cause an increase in PKC activity. This increase begins 1 hr after conditioning, lasts up to 3 d, and is attributable to an increased level of constitutive PKC. The increased level of constitutive PKC consists of an early proteolysis-dependent phase and a late phase that requires RNA and protein synthesis. Whereas constitutive activity is marginal in untrained animals (<5% of basal calcium-dependent PKC activity), three-trial conditioning produces an at least 600% increase in constitutive activity, as compared with that in untrained animals. This increase is similar at 3 and 18 hr after conditioning and corresponds to the learning-induced elevation of PKC activity measured in the presence of calcium and DAG. Hence, the constitutive PKC activity may be fully responsible for the long-lasting PKC activation induced by conditioning. Whereas the transient activation of PKC induced by single or paired stimuli is blocked in the presence of the PKC inhibitor Gö 7874, long-lasting activation 3 and 18 hr after conditioning is not susceptible to this inhibition. Thus, the calcium independence and the lack of inhibition by Gö 7874 support the hypothesis that long-lasting and transient PKC activation are mediated by different mechanisms (Grunbaum, 1998).

Because the direct detection of PKM is not possible, the in vivo effect of protease inhibitors on conditioning-induced PKC activation was investigated. The formation of PKM probably is mediated by the thiol protease calpain. Calpain homologs have been purified from honeybee brains and can be inhibited by the thiol protease inhibitor E 64. E 64 was injected before conditioning, because the induction of a proteolytic mechanism in LTP was described as taking place during or shortly after training. Elevation of PKC activity 3 hr after conditioning was blocked in E 64-treated bees, as compared with that in PBS-injected control animals. However, 18 hr after conditioning, PKC activation in E 64-treated bees was not distinguishable from that in PBS-injected controls. PKC activity in untrained animals was not changed by E 64. Hence, a proteolytic mechanism is required for the induction of PKC activation during the first hour after conditioning. To investigate the contribution of gene expression to PKC activation after conditioning, transcription and translation inhibitors were tested for their ability to interfere with PKC activation in vivo. It was assumed that RNA and protein synthesis are not required during, but after, training. Therefore, inhibitors of RNA (actinomycin D) and protein synthesis (a mixture of cycloheximide and anisomycin) were injected 1 hr after training. Actinomycin D does not interfere with PKC activation 3 hr after conditioning. However, 18 hr after conditioning, no activation of PKC was measured in bees treated with transcription or translation inhibitors. Taken together, these data suggest that the early phase of conditioning-induced PKC activation is dependent on proteolysis and is not required for the induction of the late phase of conditioning-induced PKC activation, which requires the synthesis of RNA and proteins. Thus Inhibition of the pathways resulting in constitutive PKC selectively impairs distinct phases of multiple-trial induced memory. The inhibition of the proteolytic mechanism has an instant effect on an early phase of multiple-trial induced memory but does not affect acquisition and the late phase of memory. Blocking of the transient PKC activation during conditioning does not affect the induction of memory formation. Thus, the constitutive PKC in the antennal lobe seems to contribute to the early phase of memory that is induced by multiple-trial conditioning (Grunbaum, 1998).