Experimental approaches to understanding the role of protein phosphorylation in the regulation of neuronal function

Studies by Earl Sutherland and his colleagues on hormonal regulation of the breakdown of glycogen in liver resulted in the discovery that the first step in the action of many hormones is to increase the synthesis of cAMP by activating adenylate cyclase (Raft et al 1957, Sutherland & Rall 1958, Robison et al 1968). It was later established that cAMP exerts its effects by stimulating protein kinases that catalyze the phosphorylation of specific functional proteins and thereby regulate their activity (Walsh et al 1968, Kuo & Greengard 1969, Krebs & Beavo 1979). The discovery that the brain contains a high concentration of cAMP-dependent protein kinase led to the proposal that protein phosphorylation might play an important role in regulation of neuronal properties by neurotransmitters and neurohormones (Miyamoto et al 1969). In particular, it seemed that protein phosphorylation, which usually takes place on a time scale of hundreds of milliseconds or longer, might be a mechanism underlying relatively long-lasting changes in neuronal properties such as "slow" changes in post-synaptic potentials (McAfee & Greengard 1972), changes in the rate of transmitter synthesis (Morgenroth et al 1975), or changes in gene expression (Klein & Berg 1970). The biochemists and neurobiologists who took up the study of brain protein phosphorylation hoped to gain insight into some of the mechanisms underlying changes in neuronal excitability and synaptic efficacy and also, perhaps, into processes that govern the development of various neuronal types during the formation of the nervous system. This line of research was bolstered by the findings that the brain contains not only high concentrations of protein kinases, but also protein phosphatases, adenylate cyclase, and phosphodiesterase (Greengard 1976), and also by the discovery that several neurotransmitters stimulate the synthesis of second messengers such as cyclic AMP and cyclic GMP by binding to specific receptors on the surfaces of neurons (for reviews see Nathanson 1977, Greengard 1981)

Characterization of the extracellular lipase of Bacillus subtilis and its relationship to a membrane-bound lipase found in a mutant strain

Bacillus subtilis CMK33 is a mutant that is more osmotically fragile than the wild type when it is converted to the protoplast form. The protoplasts of this mutant contain a membrane-bound lipase, which is not found in protoplasts of the wild type. Hydrolysis of the membrane lipid of mutant protoplasts by the lipase is the cause of their fragility. A protein found in the wild type organism specifically inhibits the lipase (Kent, C., and Lennarz, W. J. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 2793-2797). This paper reports that cultures of both mutant and wild type cells contain an extracellular lipase which accumulates during the logarithmic phase of growth. The extracellular activity appears to be induced by a component of the growth medium. The membrane-bound lipase of the mutant has been partially purified and its properties have been compared to those of the extracellular lipase of the wild type. Their properties and sensitivity to the wild type inhibitor are similar, which suggests that the two molecules are closely related. The subcellular location of the lipase in the mutant has been investigated and compared to the location of the membrane-bound portion of the lipase inhibitor in the wild type. The lipase is located almost exclusively in the cytoplasmic membrane and not in mesosomal vesicles. In contrast, the lipase inhibitor is located in both types of membranes and is more concentrated in mesosomal vesicles. Under appropriate conditions, the appearance of new extracellular lipase activity in mutant cultures is paralleled by the loss of an equivalent amount of lipase activity from protoplasts prepared from the cells. This suggests that the membrane-bound lipase may be an intermediate in the secretion of the extracellular lipase. Because of the mutation in B. subtilis CMK33, which results in the absence of the lipase inhibitor, this intermediate can be found in protoplasts of the mutant, although it is not detectable in the wild type. Consequently, the mutant may be useful in studies of the mechanism of secretion of exoenzymes by Bacilli

Mammalian Septins Nomenclature

There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1–SEPT10 and Sept1–Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase “v” and a number, e.g., SEPT4_v1

Regional distribution of type II Ca^(2+)/calmodulin-dependent protein kinase in rat brain

The distribution of type II Ca^(2+)/calmodulin-dependent protein kinase has been mapped in rat brain by immunochemical and immunohistochemical methods using an antibody against its alpha-subunit. The concentration of the kinase, measured by radioimmunoassay, varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of the total hippocampal protein, 1.3% of cortical protein, and 0.7% of striatal protein. It is less concentrated in lower brain structures, ranging from about 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla. The gradient of staining intensity observed in brain sections by immunohistochemistry corroborates this distribution. Neurons and neuropil of the hippocampus are densely stained, whereas little staining is observed in lower brain regions such as the superior colliculus. Within the diencephalon and midbrain, dense staining is observed only in thalamic nuclei and the substantia nigra. The skewed distribution of alpha-subunit appears to be due in part to the occurrence in the cerebellum and pons/medulla of forms of the kinase with a high ratio of beta- to alpha-subunits. However, most of the variation is due to the extremely high concentration of the kinase in particular neurons, especially those of the hippocampus, cortex and striatum. The unusually high expression of the kinase in these neurons is likely to confer upon them specialized responses to calcium ion that are different from those of neurons in lower brain regions

Distinct forebrain and cerebellar isozymes of type II Ca^(2+)/calmodulin-dependent protein kinase associate differently with the postsynaptic density fraction

Forebrain and cerebellar Type II Ca2+/calmodulin-dependent protein kinases have different subunit compositions. The forebrain holoenzyme, characterized in our laboratory, is a 650-kDa holoenzyme composed of 50-kDa alpha-subunits and 60-kDa beta-subunits assembled in approximately a 3:1 ratio (Bennett, M. K., Erondu, N. E., and Kennedy, M. B. (1983) J. Biol. Chem. 258, 12735-12744). The cerebellar isozyme is a 500-kDa holoenzyme composed of alpha-subunits and beta-subunits assembled in almost the converse ratio, approximately four beta-subunits for each alpha-subunit. When compared by tryptic peptide mapping and by immunochemical techniques, the beta-subunits from the two brain regions are indistinguishable and the alpha-subunits appear closely related. The specific activities, substrate specificities, and catalytic constants of the cerebellar and forebrain isozymes are similar, suggesting that the alpha- and beta-subunits contain similar catalytic sites. However, two differences in the properties of the isozymes may result in functional differences between them in vivo. First, the apparent affinity of the cerebellar kinase for Ca2+/calmodulin is 2-fold higher than that of the forebrain kinase. Second, the two isozymes appear to associate differently with subcellular structures. Approximately 85% of the cerebellar kinase and 50% of the forebrain kinase remain in the particulate fraction after homogenization under standard conditions. However, they are present in different amounts in postsynaptic density fractions. Postsynaptic densities prepared from forebrain contain the forebrain isozyme. Immunochemical measurements show that it comprises approximately 16% of their total protein. In contrast, postsynaptic densities prepared from cerebellum contain the cerebellar isozyme, but it comprises only approximately 1-2% of their total protein. Thus, the alpha-subunit may play a role in anchoring Type II Ca2+/calmodulin-dependent protein kinase to postsynaptic densities

Deduced Primary Structure of the β Subunit of Brain Type II Ca2+/calmodulin-dependent Protein Kinase Determined by Molecular Cloning

cDNA clones coding for the β subunit of rat brain type II Ca2+/calmodulin-dependent protein kinase were isolated and sequenced. The clones, including one containing the entire coding region, hybridize at high stringency to a single band of poly(A)+ RNA of length 4.8 kilobases. The subunit coded for by the clones was identified by in vitro transcription of the cDNA followed by translation of the resulting RNA. The DNA sequence of the clones contains a single long open reading frame (1626 nucleotides) coding for a protein of 542 amino acids with a molecular weight of 60,333, the amino-terminal half of which is homologous to several other protein kinases. Potential ATP- and calmodulin-binding domains were identified. Two independent clones contain an identical 45-nucleotide deletion, relative to the clones described above, resulting in a shorter open reading frame coding for a protein of molecular weight 58,000. This suggests that the minor, 58-kDa β' subunit of the type II Ca2+/calmodulin-dependent kinase may be synthesized on a separate message

Detailed state model of CaMKII activation and autophosphorylation

By combining biochemical experiments with computer modelling of biochemical reactions we elucidated some of the currently unresolved aspects of calcium-calmodulin-dependent protein kinase II (CaMKII) activation and autophosphorylation that might be relevant for its physiological function and provided a model that incorporates in detail the mechanism of CaMKII activation and autophosphorylation at T286 that is based on experimentally determined binding constants and phosphorylation rates. To this end, we developed a detailed state model of CaMKII activation and autophosphorylation based on the currently available literature, and constrained it with data from CaMKII autophosphorylation essays. Our model takes exact phosphorylation patterns of CaMKII holoenzymes into account, and is valid at physiologically relevant conditions where the concentrations of calcium and calmodulin are not saturating. Our results strongly suggest that even when bound to less than fully calcium-bound calmodulin, CaMKII is in the active state, and indicate that the autophosphorylation of T286 by an active non-phosphorylated CaMKII subunit is significantly faster than by an autophosphorylated CaMKII subunit. These results imply that CaMKII can be efficiently activated at significantly lower calcium concentrations than previously thought, which may explain how CaMKII gets activated at calcium concentrations existing at synapses in vivo. We also investigated the significance of CaMKII holoenzyme structure on CaMKII autophosphorylation and obtained estimates of previously unknown binding constants

Regulation of the Neuron-specific Ras GTPase-activating Protein, synGAP, by Ca2+/Calmodulin-dependent Protein Kinase II

synGAP is a neuron-specific Ras GTPase-activating protein found in high concentration in the postsynaptic density fraction from mammalian forebrain. Proteins in the postsynaptic density, including synGAP, are part of a signaling complex attached to the cytoplasmic tail of the N-methyl-D-aspartate-type glutamate receptor. synGAP can be phosphorylated by a second prominent component of the complex, Ca2+/calmodulin-dependent protein kinase II. Here we show that phosphorylation of synGAP by Ca2+/calmodulin-dependent protein kinase II increases its Ras GTPase-activating activity by 70-95%. We identify four major sites of phosphorylation, serines 1123, 1058, 750/751/756, and 764/765. These sites together with other minor phosphorylation sites in the carboxyl tail of synGAP control stimulation of GTPase-activating activity. When three of these sites and four other serines in the carboxyl tail are mutated, stimulation of GAP activity after phosphorylation is reduced to 21 ± 5% compared with 70-95% for the wild type protein. We used phosphosite-specific antibodies to show that, as predicted, phosphorylation of serines 765 and 1123 is increased in cultured cortical neurons after exposure of the neurons to the agonist N-methyl-D-aspartate

Biochemistry and neuroscience: the twain need to meet

Neuroscience has come to mean the study of electrophysiology of neurons and synapses, micro and macro-scale neuroanatomy, and the functional organization of brain areas. The molecular axis of the field, as reflected in textbooks, often includes only descriptions of the structure and function of individual channels and receptor proteins, and the extracellular signals that guide development and repair. Studies of cytosolic ‘molecular machines’, large assemblies of proteins that orchestrate regulation of neuronal functions, have been neglected. However, a complete understanding of brain function that will enable new strategies for treatment of the most intractable neural disorders will require that in vitro biochemical studies of molecular machines be reintegrated into the field of neuroscience

Biochemistry and neuroscience: the twain need to meet

Neuroscience has come to mean the study of electrophysiology of neurons and synapses, micro and macro-scale neuroanatomy, and the functional organization of brain areas. The molecular axis of the field, as reflected in textbooks, often includes only descriptions of the structure and function of individual channels and receptor proteins, and the extracellular signals that guide development and repair. Studies of cytosolic ‘molecular machines’, large assemblies of proteins that orchestrate regulation of neuronal functions, have been neglected. However, a complete understanding of brain function that will enable new strategies for treatment of the most intractable neural disorders will require that in vitro biochemical studies of molecular machines be reintegrated into the field of neuroscience