pH-sensitive, Ca2+/calmodulin-dependent phosphorylation of unique protein in molluscan nervous system

Gillette, Rhanor; Gillette, Martha L; Lipeski, Lauren; Connor, James R.

doi:10.1016/0304-4165(90)90036-v

Cited by 5 publications

(5 citation statements)

References 17 publications

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“…eEF-2 Phosphorylation in LiVer Tissue Extracts. Our studies of the effect of pH on eEF-2 phosphorylation was prompted by the report of Gillette et al (38), who found that acidic pH selectively stimulated phosphorylation of a protein with properties similar to eEF-2 in mollusk neuron extracts. It had been shown previously that phosphorylated eEF-2 in liver extracts can be easily identified as a major phosphorylated band (39).…”

Section: Resultsmentioning

confidence: 99%

Regulation of Elongation Factor-2 Kinase by pH

et al. 2002

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Elongation factor-2 kinase (eEF-2K) is a Ca(2+)/calmodulin-dependent protein kinase that phosphorylates and inactivates eEF-2 and that can regulate the rate of protein synthesis at the elongation stage. Here we report that a slight decrease in pH, within the range observed in vivo, leads to a dramatic activation of eEF-2K. The activity of eEF-2K in mouse liver extracts, as well as the activity of purified recombinant human eEF-2K, is low at pH 7.2-7.4 and is increased by severalfold when the pH drops to 6.6-6.8. eEF-2K requires calmodulin for activity at neutral as well as acidic pH. Kinetic studies demonstrate that the pH does not affect the K(M) for ATP or eEF-2 and activation of eEF-2K at acidic pH is due to an increase in V(max). To analyze the potential role of eEF-2K in regulating protein synthesis by pH, we constructed a mouse fibroblast cell line that expresses eEF-2K in a tetracycline-regulated manner. Overexpression of eEF-2K led to a decreased rate of protein synthesis at acidic pH, but not at neutral pH. Our results suggest that pH-dependent activation of eEF-2K may play a role in the global inhibition of protein synthesis during tissue acidosis, which accompanies such processes as hypoxia and ischemia.

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Section: Resultsmentioning

confidence: 99%

Regulation of Elongation Factor-2 Kinase by pH

et al. 2002

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“…Taking the bit in our teeth, we undertook a survey of pH-sensitive phosphorylation in proteins separated on 1- and 2-D gels. 23 The findings were interesting: of around 200 separable phosphoproteins, only a single one’s phosphorylation state was quite sensitive to incubation of ganglion homogenate over a physiological pH range of 7.0-7.4. This protein was also identifiable in homogenates from lobster, sea-urchin, and earthworm.…”

Section: Modeling and Measuring Camp Actionsmentioning

confidence: 99%

“…We virtually ignored recent findings of cyclic nucleotide-gated cation channels in retina and olfactory epithelium. Taking the bit in our teeth, we undertook a survey of pH-sensitive phosphorylation in proteins separated on 1- and 2-D gels . The findings were interesting: of around 200 separable phosphoproteins, only a single one’s phosphorylation state was quite sensitive to incubation of ganglion homogenate over a physiological pH range of 7.0–7.4.…”

Section: Intracellular Phmentioning

confidence: 99%

“…The unique finding of pH sensitivity in the phosphorylation state seemed important, but we found it hard to publish, perhaps partly because the pH i sensitivity seemed a bit too exotic in the cell biology and biochemistry journals to which we submitted it. When it finally came out, we had already learned that the I Na,Camp channels were activated by direct cAMP ligand gating, and we did not wish to put more effort into the phosphorylation work, however significant it seemed at the time. Fortunately, Alexey Ryazanov learned of our work and his laboratory then found that the phosphorylating kinase, eEF-3 kinase, was exquisitely sensitive to both Ca 2+ -calmodulin and pH .…”

Section: Intracellular Phmentioning

confidence: 99%

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cAMP, Ca²⁺, pH_i, and NO Regulate H-like Cation Channels That Underlie Feeding and Locomotion in the Predatory Sea Slug Pleurobranchaea californica

Green

Huang

Sudlow

et al. 2018

ACS Chem. Neurosci.

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A systems approach to regulation of neuronal excitation in the mollusc Pleurobranchaea has described novel interactions of cyclic AMP-gated cation current (I), Ca, pH, and NO. I appears in many neurons of feeding and locomotor neuronal networks. It is likely one of the family of hyperpolarization-activated, cyclic-nucleotide-gated currents (h-current) of vertebrate and invertebrate pacemaker networks. There are two isoforms. Ca regulates both voltage dependence and depolarization-sensitive inactivation in both isoforms. The Type 1 I of the feeding network is enhanced by intracellular acidification. A direct dependence of I on cAMP allows the current to be used as a reporter on cAMP concentrations in the cell, and from there to the intrinsic activities of the synthetic adenyl cyclase and the degradative phosphodiesterase. Type 2 I of the locomotor system is activated by serotonergic inputs, while Type 1 of the feeding network is thought to be regulated peptidergically. NO synthase activity is high in the CNS, where it differs from standard neuronal NO synthase in not being Ca sensitive. NO acidifies pH, potentiating Type 1, and may act to open proton channels. A cGMP pathway does not mediate NO effects as in other systems. Rather, nitrosylation likely mediates its actions. An integrated model of the action of cAMP, Ca, pH, and NO in the feeding network postulates that NO regulates proton conductance to cause neuronal excitation in the cell body on the one hand, and relief of activity-induced hyperacidification in fine dendritic processes on the other.

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“…For intracellular pressure injection, protein kinase inhibitor (PKI) protein (bovine heart type II from Sigma) was suspended in 106 rnh KC1 and 10 mM HEPES at DH 7.3. dialvzed for 24 hr aeainst the same buffer and diluted to a finalconcentration of 14.1 mg/A. This concentration of the specific batch we used effectively inhibited cyclic AMPdependent phosphorylation in Pleurobrunchaea nervous system homogenates by more than 80% (Gillette et al, 1990). The synthetic, 20 amino acid active fragment of the PKI protein was obtained in >99Oh pure form (Bachem Ltd.).…”

Section: Specimensmentioning

confidence: 99%

cAMP-activated Na+ current of molluscan neurons is resistant to kinase inhibitors and is gated by cAMP in the isolated patch

et al. 1993

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The cAMP-dependent Na+ current (INa,cAMP) modulates excitability in many molluscan neurons. Rapid activation of INa,cAMP by cyclic nucleotide, its ion dependence, and its blockade by divalent cations resemble cyclic nucleotide-activated cation currents in vertebrate photoreceptors and olfactory receptors, where activation has been found to be independent of kinase activity. We tested the phosphorylation dependence of INa,cAMP in neurons of the feeding and locomotory networks of the predatory marine snail Pleurobranchaea. Identified neurons of pedal and buccal ganglia were axotomized for recording the INa,cAMP response to iontophoretic injection of cAMP under two-electrode voltage clamp. Intracellular injections of specific peptide inhibitor of protein kinase A had no blocking effects on activation of INa,cAMP by iontophoretic injection of cAMP. Inward single-channel currents were activated in excised inside-out patches during exposure to cAMP in salines without added ATP. Sodium was the major current carrying ion. Two distinct types of INa,cAMP channel activity were observed, where opening probability and open times differed, but conductance was similar, 36.7 pS. These observations suggest that INa,cAMP activation occurs by direct binding of cAMP to a regulatory site at the channel, rather than by phosphorylation.

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pH-sensitive, Ca2+/calmodulin-dependent phosphorylation of unique protein in molluscan nervous system

Cited by 5 publications

References 17 publications

Regulation of Elongation Factor-2 Kinase by pH

Regulation of Elongation Factor-2 Kinase by pH

cAMP, Ca²⁺, pH_i, and NO Regulate H-like Cation Channels That Underlie Feeding and Locomotion in the Predatory Sea Slug Pleurobranchaea californica

cAMP-activated Na+ current of molluscan neurons is resistant to kinase inhibitors and is gated by cAMP in the isolated patch

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pH-sensitive, Ca2+/calmodulin-dependent phosphorylation of unique protein in molluscan nervous system

Cited by 5 publications

References 17 publications

Regulation of Elongation Factor-2 Kinase by pH

Regulation of Elongation Factor-2 Kinase by pH

cAMP, Ca2+, pHi, and NO Regulate H-like Cation Channels That Underlie Feeding and Locomotion in the Predatory Sea Slug Pleurobranchaea californica

cAMP-activated Na+ current of molluscan neurons is resistant to kinase inhibitors and is gated by cAMP in the isolated patch

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cAMP, Ca²⁺, pH_i, and NO Regulate H-like Cation Channels That Underlie Feeding and Locomotion in the Predatory Sea Slug Pleurobranchaea californica