Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation

Hanson, Phyllis I.; Kapiloff, Michael S.; Lou, Lillian; Rosenfeld, Michael G.; Schulman, Howard

doi:10.1016/0896-6273(89)90115-3

Cited by 275 publications

(205 citation statements)

References 45 publications

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“…In a first reaction step, immunoprecipitated CaMKII was incubated for 30 min at 30°C with 5 mM CaCl 2 and 5 μM calmodulin in 50 μl reaction mixture consisting of 50 mM HEPES pH 7.5, 10 mM MgCl 2 , 0.5 mM dithiothreitol (DTT), 2 μM CaM, 100 nM microcystin, 0.5 mM cold ATP. A 10 μl aliquot from the first reaction was than incubated with 25 mM EGTA, 0.5 mM Autocamtide 33 and 50 μM ATP [1,500 cpm/pmol (γ-32 P)ATP] in order to determine CaMKII activity on its peptide substrate Autocamtide. The reaction was performed for 30 min at 30°C and 20 μl aliquots of the reaction Figure 6.…”

Section: Disclosure Of Potential Conflicts Of Interestmentioning

confidence: 99%

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

et al. 2012

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The calcium/calmodulin-dependent kinase II (CaMKII) participates with Ras to Raf-1 activation, and it is necessary for activation of the extracellular signal-regulated kinase (ERK) by different factors in epithelial and mesenchimal cells. Raf-1 activation is a complex multistep process, and its maximal activation is achieved by phosphorylation at Y341 by Src and at S338 by other kinase/s. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 remains to be definitively identified. In this study, we verified the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To do so, we determined the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors. Serum, fibronectin, Src(Y527) and Ras(V12) activated CaMKII and ERK, at different extents. The inhibition of CaMKII attenuated Raf-1 and ERK activation by all these factors. CaMKII was also necessary for the phosphorylation of Raf-1 at S338 by serum, fibronectin and Ras. Conversely, inhibition of Pak3 activation by blocking phosphatidylinositol-3-kinase was ineffective. The direct phosphorylation of S338 Raf-1 by CaMKII was demonstrated in vitro by interaction of purified kinases. These results demonstrate that Ras activates CaMKII, which, in turn, phosphorylates Raf-1 at S338 and participates in ERK activation upon different stimuli

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Section: Disclosure Of Potential Conflicts Of Interestmentioning

confidence: 99%

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

et al. 2012

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“…This reaction has been proposed as a "molecular switch," translating transient calcium elevation into prolonged kinase activity (15, 16), which becomes subject to regulation by protein phosphatases. (19).Isolation of PSDs-PSDs were prepared from adult rat forebrains flash frozen within 45 s of euthanasia by detergent lysis of synaptosomes (20) except that 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 10 g/ml leupeptin were included in all buffers. Synaptosomes were lysed in 1% (v/v) Triton X-100 and 150 mM KCl, and a second subsequent sucrose gradient was omitted because it yielded no further purification.…”

mentioning

confidence: 99%

Translocation of Autophosphorylated Calcium/Calmodulin-dependent Protein Kinase II to the Postsynaptic Density

Strack

Choi

Lovinger

et al. 1997

Journal of Biological Chemistry

280

236

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Calcium/calmodulin-dependent protein kinase II (CaMKII) undergoes calcium-dependent autophosphorylation, generating a calcium-independent form that may serve as a molecular substrate for memory. Here we show that calcium-independent CaMKII specifically binds to isolated postsynaptic densities (PSDs), leading to enhanced phosphorylation of many PSD proteins including the ␣-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-type glutamate receptor. Furthermore, binding to PSDs changes CaMKII from a substrate for protein phosphatase 2A to a protein phosphatase 1 substrate. Translocation of CaMKII to PSDs occurs in hippocampal slices following treatments that induce CaMKII autophosphorylation and a form of long term potentiation. Thus, synaptic activation leads to accumulation of autophosphorylated, activated CaMKII in the PSD. This increases substrate phosphorylation and affects regulation of the kinase by protein phosphatases, which may contribute to enhancement of synaptic strength.CaMKII 1 isoforms comprise a family of broad specificity, calcium-activated kinases (1, 2). The ␣ and ␤ isoforms are abundantly expressed in the brain, with ␣ making up as much as 2% of total protein in certain brain regions (3). CaMKII is particularly enriched in PSDs (4, 5), cytoskeletal specializations apposed to the postsynaptic membrane of excitatory synapses that are thought to be scaffolds for neurotransmitter receptors, ion channels, and their postsynaptic modulators and effectors (reviewed in Refs. 6 and 7). Earlier reports suggested that CaMKII␣ constitutes as much as 50% of total PSD protein (8 -10), but PSDs prepared from rapidly homogenized brains are only 2-3-fold enriched in CaMKII␣ compared with whole forebrain extracts (3,11). CaMKII␣ knockout mice show impaired hippocampal long term potentiation, a cellular model for learning and memory (12). Conversely, introduction of CaMKII␣ into neurons augments postsynaptic responses and occludes further electrically induced long term potentiation (13,14).CaMKII␣ undergoes calcium/calmodulin-dependent autophosphorylation on Thr 286 in its regulatory domain, rendering the kinase partially calcium-independent (1, 2). This reaction has been proposed as a "molecular switch," translating transient calcium elevation into prolonged kinase activity (15, 16), which becomes subject to regulation by protein phosphatases. (19).Isolation of PSDs-PSDs were prepared from adult rat forebrains flash frozen within 45 s of euthanasia by detergent lysis of synaptosomes (20) except that 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 10 g/ml leupeptin were included in all buffers. Synaptosomes were lysed in 1% (v/v) Triton X-100 and 150 mM KCl, and a second subsequent sucrose gradient was omitted because it yielded no further purification. PSDs displayed typical "donut" morphology by video-enhanced differential-interference contrast microscopy (21). PSDs prepared in the absence or the presence of the protein phosphatase inhibitor microcystin-LR (1 M) contained ...

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“…The activity of the transfected CaM kinases in the cell extract was assayed using a peptide substrate autocamtide 2 as described previously (Hanson et al, 1989). Assay conditions were similar to that previously described excepting that the specific activity of the ['y-32p]ATP used was 1 Ci/mmole.…”

Section: Additional Methodsmentioning

confidence: 99%

Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus

Srinivasan¹,

Edman²,

Schulman³

1994

Trends in Cell Biology

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Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation

Cited by 275 publications

References 45 publications

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

Translocation of Autophosphorylated Calcium/Calmodulin-dependent Protein Kinase II to the Postsynaptic Density

Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus

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