Experimental Approaches to Understanding the Role of Protein Phosphorylation in the Regulation of Neuronal Function

Kennedy, Mary B.

doi:10.1146/annurev.ne.06.030183.002425

Cited by 91 publications

(26 citation statements)

References 124 publications

(63 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Such long-term changes could involve alterations in cell metabolism (e.g., ref. 36) or in genomic regulation and protein synthesis (37-39) brought about via cAMP-mediated phosphorylations (40,41). Thus, altered somatic cAMP levels may provide a biochemical link between short-and longterm plastic changes in neurons.…”

Section: Discussionmentioning

confidence: 99%

Associative conditioning analog selectively increases cAMP levels of tail sensory neurons in Aplysia.

Ocorr¹,

Walters²,

Byrne³

1985

Proc. Natl. Acad. Sci. U.S.A.

188

View full text Add to dashboard Cite

Bilateral clusters of sensory neurons in the pleural ganglia of Aplysia contain cells involved in a defensive tail withdrawal reflex. These cells exhibit heterosynaptic facilitation in response to noxious skin stimulation that can be mimicked by the application of serotonin. Recently it has been shown that this facilitation can be selectively amplified by the application of a classical conditioning procedure to individual sensory neurons. We now report that an analog of this classical conditioning paradigm produces a selective amplification of the cAMP content of isolated sensory neuron clusters. The enhancement is achieved within a single trial and appears to be localized to the sensory neurons. These results indicate that a pairing-specific enhancement of cAMP levels may be a biochemical mechanism for associative neuronal modifications and perhaps learning.Associative modifications within individual neurons have recently been implicated as mechanisms for associative learning and memory (1-5). A recently developed preparation that offers particular promise for the analysis of biochemical and biophysical modifications underlying neural plasticity is the defensive tail withdrawal reflex in the marine mollusc Aplysia. An associative conditioning paradigm applied directly to individual sensory neurons can specifically modify monosynaptic connections between these cells and the motor neurons involved in tail withdrawal (3). This was shown with a differential conditioning procedure that utilized intracellular activation of individual tail sensory cells as the conditioned stimulus (CS) and shock to the skin as the unconditioned stimulus (US). Since the US was delivered outside the receptive fields of the sensory neurons examined, these neurons were not activated by the US. However, they were exposed to diffuse neuromodulatory effects of the US that produced heterosynaptic facilitation of all of the connections of the sensory neurons (3). Paired presentation of the CS and US resulted in a significant enhancement of the excitatory postsynaptic potential (EPSP) amplitude compared to the facilitation produced when the CS and US were separated by 2 min or when the US was presented alone (3). We termed this enhancement "activity-dependent neuromodulation" because the effects of a presumed neuromodulator released by the US are specifically enhanced by spike activity that immediately precedes the US. Although models formally similar to activity-dependent neuromodulation have been proposed previously as candidate mechanisms for associative memory (6-8), these and similar results obtained independently by Hawkins et al. (4) in Aplysia provided direct experimental evidence for such a phenomenon at the cellular level.The tail withdrawal is sensitized by the same weak electrical stimulation of the skin used as the US (above), and the sensitization is accompanied by heterosynaptic facilitation of the monosynaptic connections between the sensory and motor neurons (9). This heterosynaptic facilitation can be mimicked by subs...

show abstract

Section: Discussionmentioning

confidence: 99%

Associative conditioning analog selectively increases cAMP levels of tail sensory neurons in Aplysia.

Ocorr¹,

Walters²,

Byrne³

1985

Proc. Natl. Acad. Sci. U.S.A.

188

View full text Add to dashboard Cite

show abstract

“…Further studies will be needed to elucidate the importance of this complex regulatory system. Ca 2+ -CALMODULIN-SENSITIVE PROTEIN KINASES-A number of the actions mediated by Ca 2+ and calmodulin in the nervous system appear to reflect activation of Ca 2+ -calmodulin-sensitive protein kinases with restricted substrate specificities (355). In contrast to cAMP-dependent protein kinases, which contain only a single catalytic subunit, Ca 2+ -calmodulin kinases exist as several distinct species with specific targets whose diversity is only now beginning to be explored.…”

Section: Calmodulin Calcineurin Interactions-calmodulin the Virtuallmentioning

confidence: 99%

A Molecular Description of Nerve Terminal Function

Reichardt¹,

Kelly²

1983

Annu. Rev. Biochem.

226

View full text Add to dashboard Cite

The nerve terminal is a specialized region of a neuron, separated from the neuronal soma by an axon that can be exceedingly long, whose function is to release neurotransmitter when stimulated by an electrical signal carried by the axon. In this review, we describe the enzymes, channels, and other proteins presently thought to be important in nerve terminal function. Recent progress in the area has been so dramatic that it is becoming possible to relate simple changes in behavior to modifications of defined proteins in particular nerve terminals.Neurotransmitters are stored in synaptic vesicles and are released by fusion of these vesicles to the plasma membrane. Vesicle fusion is triggered by Ca 2+ -influx through specific Ca 2+ channels that open in response to depolarization of the plasma membrane and is terminated by the disappearance of Ca 2+ from the vicinities of the active zones. The voltage signal that opens the Ca 2+ gates is not constant, but also subject to regulation. The key elements are the Na + and K + channels in the nerve terminal. These channels are localized to distinct regions of many neurons and different neurons have different quantities of the different channel types. The voltage sensitive Na + channel is responsible for depolarizing the membrane. This channel has recently been purified and reconstituted in artificial membranes, so many of its properties are known. K + channels are responsible for repolarizing the membrane. Several K + channels have been identified: some activated by voltage, some by intracellular Ca 2+ and others by neurotransmitters. The properties of several of these have recently been shown to be altered by cAMP-dependent protein kinases, resulting in long-term changes in neuronal activity and the efficiency of synaptic transmission. The voltage-dependent Ca 2+ channels can also be modified by cAMP-dependent protein kinases. Many of the neurotransmitters and hormones that modulate the efficiency of transmitter release apparently do so by modifying the Ca 2+ channels. Much of the recent progress in molecular studies on the K + and Ca 2+ channels has been made possible by a dramatic new technique called "patch clamping" that permits individual ion channels to be examined on the tip of a microelectrode. In many ways this technology circumvents the need for biochemical isolation and reconstitution.Maintenance of the Na + and K + concentrations in neurons requires the classical Na, + K + -ATPase that is found in all cell types. Recent experiments suggest that a novel form of this enzyme exists in neural tissues. This pump appears to be localized to anatomically and functionally distinct regions of many neurons. To restore the cytoplasmic Ca 2+ concentrations to original levels, the nerve terminal has an array of Ca 2+ removal systems including a Na + :Ca 2+ antiporter in the plasma membrane, a Ca 2+ porter in the mitochondrial inner membrane and several distinct ATP-dependent Ca 2+ uptake systems in the plasma membrane, smooth endoplasmic reticulum, and synaptic vesicles.The...

show abstract

“…Recently, the presence of ecto-protein kinase activity at the surface of neuronal cells has been demonstrated, and specific phosphorylated substrates of the neuronal ecto-protein kinase have been identified (Ehrlich et al, 1986(Ehrlich et al, , 1990Tsuji et al, 1988;Zhang et al, 1988). The identification, localization, and characterization of specific endogenous protein substrates for phosphorylation activity are important for determining the physiological function of various protein kinases (Kennedy, 1983;Ehrlich, 1984Ehrlich, , 1987. Thus, the identification and characterization of the specific surface membrane proteins phosphorylated by extracellular ATP should provide insight into the mechanisms by which ecto-protein kinases are involved in the regulation of various neuronal functions (Ehrlich et al, 1986Ehrlich, 1987;Hendley et al, 1988;Muramoto et al, 1988;Hardwick et al, 1989).…”

mentioning

confidence: 99%

Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Pawłowska

Hogan

Kornecki

et al. 1993

Journal of Neurochemistry

View full text Add to dashboard Cite

Abstract:The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [y-32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32P,. This activity WBS abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P,. As also expected from ecto-protein kinase activity, PC 12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-a-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as e.wIusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75-87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca2+-dependent, high-affinity norepinephrine uptake in PC 12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra-and extracellular protein phosphorylation in intact PC 12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10-100 p h f ) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis. Key Words: Protein phosphorylationEcto-protein kinase-Surface phosphoproteins-ExtracelMar ATP-Nerve growth factor...

show abstract

Experimental Approaches to Understanding the Role of Protein Phosphorylation in the Regulation of Neuronal Function

Cited by 91 publications

References 124 publications

Associative conditioning analog selectively increases cAMP levels of tail sensory neurons in Aplysia.

Associative conditioning analog selectively increases cAMP levels of tail sensory neurons in Aplysia.

A Molecular Description of Nerve Terminal Function

Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Contact Info

Product

Resources

About