Calcium/calmodulin-dependent protein phosphorylation in the nervous system of Aplysia

Abstract

An afterdischarge in the bag cell neurons of Aplysia was previously shown to be associated with calcium entry into these cells and with changes in the phosphorylation state of at least two bag cell proteins (BC-I and BC-II). We have now investigated the role of calcium plus calmodulin (Cal CaM) in the control of phosphorylation of Aplysia nervous system proteins, including those of the bag cell neurons.In cell-free preparations of Aplysia CNS, we demonstrated Ca/CaM-stimulated protein phosphorylation that could be inhibited by the calmodulin-blocking drugs R24571, trifluoperazine, chlorpromazine, and W7. A number of substrate proteins for Ca/CaM-dependent protein phosphorylation with M, values from 17,000 to 310,000 were consistently observed in homogenates of the Aplysia CNS. In the bag cells, we found that a major substrate for Ca/CaM-dependent protein phosphorylation was the bag cell-specific, M, = 21,000 protein (BC-II). BC-I (M, = 33,000), on the other hand, appeared not to be a substrate for a Ca/CaM-dependent protein kinase.We found that there are a minimum of two Ca/CaM-dependent protein kinases in the Aplysia nervous system. These enzymes were distinguished on the basis of their subcellular distribution and their ability to phosphorylate distinct sites on synapsin I, an exogenous neuronal protein from vertebrates. Phosphorylation by one of these kinases (calmodulin kinase I) was on a site recovered in an M, = 10,000 proteolytic fragment of synapsin I, and phosphorylation by the other (calmodulin kinase II) was on a site recovered in an M, = 30,000 fragment. The predominant enzyme in the Aplysia CNS, as in the mammalian nervous system, was calmodulin kinase II.In addition, we compared an M, = 51,000 Aplysia substrate for Ca/CaM-dependent phosphorylation with the M, = 50,000 to 51,000 subunit of mammalian calmodulin kinase II. Both proteins showed immunoreactivity with monoclonal antibodies raised against rat calmodulin kinase II, both bound calmodulin, and phosphorylation of both proteins followed by partial proteolysis with Staphylococcus aureus protease V8 led to similar phosphopeptide maps. Our results indicate that the major form of Ca/CaM-dependent protein kinase in Aplysia CNS is homologous to mammalian calmodulin kinase II. The findings also raise the possibility that calcium/ calmodulin-dependent phosphorylation may mediate some of the long-lasting effects of intracellular calcium entry during an afterdischarge of the bag cell neurons.Calcium plays a dual role within nerve cells. It carries electrical current through voltage-gated channels in the membrane, and it also acts as a second messenger within

Calmodulin-dependent multifunctional protein kinase in Aspergillus nidulans.

“…Few detailed kinetic and substrate specificity studies have been reported comparing mammalian CaM-PK I1s to each other (32) and none has been reported comparing mammalian CaM-PK Hs to those from nonmammalian sources. DeRiemer et al reported rates of phosphorylation of synapsin I and MAP-2 of 0.89 and 0.04 nmol per min per mg of Aplysia CaM-PK II, respectively (41), which are far below those reported here for ACMPK. The isolation of a fungal multifunction Ca2+ /CaM-dependent protein kinase capable of recognizing mammalian protein substrates and being regulated by mammalian CaM indicates a high degree of functional conservation among CaM-dependent multifunctional protein kinases in eukaryotic organisms.…”

“…Multifunctional CaM-PK Ils have been isolated from mammalian brain (3,4), liver(5), pancreas (37), and skeletal muscle (38) as well as the nonmammalian tissues Torpedo electric organ (6), Aplysia neurons (7), and sea urchin egg (39). Cross-reactivity between antibodies to rat brain CaM-PK II and CaM-PK Ils from rabbit skeletal muscle (40) and Aplysia californica (41) have been reported.…”

Identification of specific mRNAs affected by treatments producing long-term facilitation in Aplysia.

“…Further study is n e e d e d to precisely define the time course of the change in expression of CaM mRNA, as well as the time course of expression for the other mRNAs. CaM can influence a variety of cell functions (for review, see Means 1988;Means et al 1991;Bachs et al 1994;Gnegy 1995), and numerous CaM-regulated enzymes have b e e n found in Aplysia neurons (Novak-Hofer and Levitan 1983;Saitoh andSchwartz 1983, 1985;DeRiemer et al 1984;Kramer et al 1988;Eliot et al 1989;Abrams et al 1991;Endo et al 1992;K.A. Ocorr and J.H.…”

Calcium/Calmodulin-Dependent Protein Kinase II

“…Studies from a number of laboratories have demonstrated that isoenzymes of CaM-kinase II are also present in most mammalian tissues examined. Indeed, the kinase has been highly purified from mammalian brain [7,[10][11][12][13][14], liver [8,15,16], skeletal muscle [17], heart [18], pancreas [19], retina [20], lung [21], parathyroid [22], mammary gland [23] and intestinal brush border [24], as well as neural tissues of Aplysia [25], electric eel [26], squid [27] and Drosophila [28]. The isoenzymes are characterized by a large native molecular mass (300-700 kDa), subunits in the range 50-62 kDa, and similar or identical catalytic properties against several protein substrates (references listed above and [29]).…”