Ca2+- and calmodulin-dependent protein phosphorylation in rat lacrimal gland.
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ABSTRACT:
SUBMITTER: Dartt DA
PROVIDER: S-EPMC1158179 | biostudies-other | 1982 Mar
REPOSITORIES: biostudies-other
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Project description:Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a serine/threonine-directed kinase that is activated following increases in intracellular Ca(2+). CaMKKβ activates Ca(2+)/calmodulin-dependent protein kinase I, Ca(2+)/calmodulin-dependent protein kinase IV, and the AMP-dependent protein kinase in a number of physiological pathways, including learning and memory formation, neuronal differentiation, and regulation of energy balance. Here, we report the novel regulation of CaMKKβ activity by multisite phosphorylation. We identify three phosphorylation sites in the N terminus of CaMKKβ, which regulate its Ca(2+)/calmodulin-independent autonomous activity. We then identify the kinases responsible for these phosphorylations as cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). In addition to regulation of autonomous activity, we find that phosphorylation of CaMKKβ regulates its half-life. We find that cellular levels of CaMKKβ correlate with CDK5 activity and are regulated developmentally in neurons. Finally, we demonstrate that appropriate phosphorylation of CaMKKβ is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKKβ-dependent signaling cascades.
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Project description:PurposeTo isolate and characterize cultured myoepithelial cells (MECs) from rat lacrimal gland and determine which purinergic receptor subtypes are present and functional in MECs.MethodsRat lacrimal glands were subjected to collagenase digestion, and MECs were grown. RT-PCR was performed for the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13) on RNA isolated from the MECs. Immunofluorescence experiments were performed with antibodies against MEC markers and P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Proteins from MECs were separated using Western blot analysis techniques. In addition, cells were incubated with Fura 2 tetra acetoxymethyl ester, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined in response to P2 purinergic agonists.ResultsMECs expressed the MEC proteins α-smooth muscle actin, vimentin, α-actinin, and adenylyl cyclase II. RT-PCR, Western blot, and immunofluorescence techniques demonstrated the presence of the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13). The purinergic agonists ATP, benzoylbenzoyl ATP (BzATP), α,β methylene ATP, UTP, 2-methylthioATP (MeSATP), and ATPγS increased [Ca(2+)](i). As BzATP binds to the P2X(7) receptor, specific characteristics of this receptor were investigated. Neither inhibitors of P2X(7) receptors nor removal of extracellular Mg(2+) or Ca(2+) had an effect on the BzATP-stimulated increase in [Ca(2+)](i). Repeated applications of BzATP desensitized this response. Inhibitors for P2Y(1), P2Y(11), and P2Y(13) each decreased the BzATP-stimulated increase in [Ca(2+)](i) with the P2Y(1) inhibitor most effective.ConclusionsMECs can be isolated from rat lacrimal glands, and they express P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Surprisingly, BzATP binds the P2Y(1) receptor, which is primarily responsible for the BzATP-stimulated increase in [Ca(2+)](i).
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Project description:PurposeAnti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers.MethodsTo address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin).ResultsHistological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased.ConclusionsThese findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.
| S-EPMC3272056 | biostudies-literature
Project description:Ca(v)1 (L-type) channels and calmodulin-dependent protein kinase II (CaMKII) are key regulators of Ca(2+) signaling in neurons. CaMKII directly potentiates the activity of Ca(v)1.2 and Ca(v)1.3 channels, but the underlying molecular mechanisms are incompletely understood. Here, we report that the CaMKII-associated protein densin is required for Ca(2+)-dependent facilitation of Ca(v)1.3 channels. While neither CaMKII nor densin independently affects Ca(v)1.3 properties in transfected HEK293T cells, the two together augment Ca(v)1.3 Ca(2+) currents during repetitive, but not sustained, depolarizing stimuli. Facilitation requires Ca(2+), CaMKII activation, and its association with densin, as well as densin binding to the Ca(v)1.3 alpha(1) subunit C-terminal domain. Ca(v)1.3 channels and densin are targeted to dendritic spines in neurons and form a complex with CaMKII in the brain. Our results demonstrate a novel mechanism for Ca(2+)-dependent facilitation that may intensify postsynaptic Ca(2+) signals during high-frequency stimulation.
| S-EPMC2990970 | biostudies-literature
Project description:During the acquisition of memories, influx of Ca2+ into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca2+influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity,including Ca2+/calmodulin-dependent protein kinase II (CaMKII), are regulated by calmodulin, a small protein that can bindup to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime,supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic models of postsynaptic signal transduction during learning.
| S-EPMC2820514 | biostudies-literature
Project description:AimsCalmodulin (CaM) regulates Na+ channel gating through binding to an IQ-like motif in the C-terminus. Ca2+/CaM-dependent protein kinase II (CaMKII) regulates Ca2+ handling, and chronic overactivity of CaMKII is associated with left ventricular hypertrophy and dysfunction and lethal arrhythmias. However, the acute effects of Ca2+/CaM and CaMKII on cardiac Na+ channels are not fully understood.Methods and resultsPurified Na(V)1.5-glutathione-S-transferase fusion peptides were phosphorylated in vitro by CaMKII predominantly on the I-II linker. Whole-cell voltage-clamp was used to measure Na+ current (I(Na)) in isolated guinea-pig ventricular myocytes in the absence or presence of CaM or CaMKII in the pipette solution. CaMKII shifted the voltage dependence of Na+ channel availability by approximately +5 mV, hastened recovery from inactivation, decreased entry into intermediate or slow inactivation, and increased persistent (late) current, but did not change I(Na) decay. These CaMKII-induced changes of Na+ channel gating were completely abolished by a specific CaMKII inhibitor, autocamtide-2-related inhibitory peptide (AIP). Ca2+/CaM alone reproduced the CaMKII-induced changes of I(Na) availability and the fraction of channels undergoing slow inactivation, but did not alter recovery from inactivation or the magnitude of the late current. Furthermore, the CaM-induced changes were also completely abolished by AIP. On the other hand, cAMP-dependent protein kinase A inhibitors did not abolish the CaM/CaMKII-induced alterations of I(Na) function.ConclusionCa2+/CaM and CaMKII have distinct effects on the inactivation phenotype of cardiac Na+ channels. The differences are consistent with CaM-independent effects of CaMKII on cardiac Na+ channel gating.
| S-EPMC2802203 | biostudies-literature
Project description:Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), the most abundant kinase at the postsynaptic density (PSD), is expected to be involved in activity-induced regulation of synaptic properties. CaMKII is activated when it binds calmodulin in the presence of Ca(2+) and, once autophosphorylated on T-286/7, remains active in the absence of Ca(2+) (autonomous form). In the present study we used a quantitative mass spectrometric strategy (iTRAQ) to identify sites on PSD components phosphorylated upon CaMKII activation. Phosphorylation in isolated PSDs was monitored under conditions where CaMKII is: (1) mostly inactive (basal state), (2) active in the presence of Ca(2+), and (3) active in the absence of Ca(2+). The quantification strategy was validated through confirmation of previously described autophosphorylation characteristics of CaMKII. The effectiveness of phosphorylation of major PSD components by the activated CaMKII in the presence and absence of Ca(2+) varied. Most notably, autonomous activity in the absence of Ca(2+) was more effective in the phosphorylation of three residues on SynGAP. Several PSD scaffold proteins were phosphorylated upon activation of CaMKII. The strategy adopted allowed the identification, for the first time, of CaMKII-regulated sites on SAPAPs and Shanks, including three conserved serine residues near the C-termini of SAPAP1, SAPAP2, and SAPAP3. Involvement of CaMKII in the phosphorylation of PSD scaffold proteins suggests a role in activity-induced structural re-organization of the PSD.
| S-EPMC2812614 | biostudies-literature
Project description:Calcium/calmodulin-dependent protein kinase II (CaMKII) is highly concentrated in the brain where its activation by the Ca2+ sensor CaM, multivalent structure, and complex autoregulatory features make it an ideal translator of Ca2+ signals created by different patterns of neuronal activity. We provide direct evidence that graded levels of kinase activity and extent of T287 (T286? isoform) autophosphorylation drive changes in catalytic output and substrate selectivity. The catalytic domains of CaMKII phosphorylate purified PSDs much more effectively when tethered together in the holoenzyme versus individual subunits. Using multisubstrate SPOT arrays, high-affinity substrates are preferentially phosphorylated with limited subunit activity per holoenzyme, whereas multiple subunits or maximal subunit activation is required for intermediate- and low-affinity, weak substrates, respectively. Using a monomeric form of CaMKII to control T287 autophosphorylation, we demonstrate that increased Ca2+/CaM-dependent activity for all substrates tested, with the extent of weak, low-affinity substrate phosphorylation governed by the extent of T287 autophosphorylation. Our data suggest T287 autophosphorylation regulates substrate gating, an intrinsic property of the catalytic domain, which is amplified within the multivalent architecture of the CaMKII holoenzyme.
| S-EPMC5748111 | biostudies-literature
Project description:Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca2+ flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca2+ effector protein that binds up to four Ca2+ ions. CaM with bound Ca2+ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca2+, a large fraction of free CaM is bound to fewer than four Ca2+ ions. Binding to targets increases the affinity of CaM's remaining Ca2+-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca2+ ions. To study CaM-dependent signaling in the spine, we designed mutant CaMs that bind Ca2+ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca2+-binding domains in the "closed" Ca2+-free conformation. We have measured their interactions with CaMKII, a major Ca2+/CaM target that mediates initiation of long-term potentiation. We show that CaM with two Ca2+ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. Our results support the idea that competition for binding of CaM with two bound Ca2+ ions may influence significantly the outcome of local Ca2+ signaling in spines and, perhaps, in other signaling pathways.
| S-EPMC1599897 | biostudies-literature
Project description:We have attempted to identify islet Ca2+/calmodulin-dependent protein kinase (CaM kinase) by comparing its activity with purified brain CaM kinase II. Islet CaM kinase, in the presence of calmodulin and Ca2+, phosphorylated major endogenous substrates of 102, 57 and 53 kDa and also exogenous glycogen synthase; brain CaM kinase II phosphorylated glycogen synthase and peptides of 57 and 53 kDa. Alloxan (1 mM) inhibited the phosphorylation of glycogen synthase and the 102, 57 and 53 kDa islet peptides by islet CaM kinase; the phosphorylation of glycogen synthase and the 57 and 53 kDa substrates by brain CaM kinase II was also inhibited by alloxan. The Ca2+ and calmodulin-dependencies of phosphorylation of the endogenous islet substrates differed. In the presence of 400 nM calmodulin, half-maximal phosphorylation was attained at Ca2+ concentrations of 80 +/- 9, 401 +/- 61 and 459 +/- 59 nM for the 102, 57 and 53 kDa substrates respectively. In the presence of 10 microM Ca2+, half-maximal phosphorylation was attained at calmodulin concentrations of 9 +/- 2, 38 +/- 2.5 and 37 +/- 2 nM for the 102, 57 and 53 kDa substrates respectively. Differential centrifugation located the 102 kDa substrate in the post-100,000 g supernatant and the 57 and 53 kDa substrates in the particulate fraction. These data suggest that islet CaM kinase is similar to, if not identical with, brain CaM kinase II, but that phosphorylation of the endogenous 102 kDa substrate occurs by a distinct kinase which shows different sensitivities to Ca2+ and calmodulin. This kinase probably corresponds to CaM kinase III and the 102 kDa peptide to elongation factor 2 (EF-2), since the 102 kDa peptide was shown to undergo ADP-ribosylation in the presence of diphtheria toxin and NAD+.
| S-EPMC1132246 | biostudies-other