Calmodulin‐dependent multifunctional protein kinase

Ca2+/calmodulin-dependent protein kinase II contains two types of subunit, a (Mr 50,000) and (3 (Mr 60,000/58,000), both of which undergo Ca2+/calmodulindependent autophosphorylation. Autophosphorylation is known to convert the enzyme to a Ca2+/calmodulin-independent form. In the present study, we have characterized the autophosphorylation sites on rat forebrain Ca2+/calmodulindependent protein kinase II that are most likely to be responsible for the generation of Ca2+/calmodulin-independence. Under coniditious (00C, low concentrations of ATP) sufficient to generate close to maximal Ca2+/calmodulin-independence, only a few of the phosphorylatable sites on the enzyme became phosphorylated. These autophosphorylation sites were examined by phospho amino acid analysis, two-dimensional thermolytic phosphopeptide mapping, and high-performance liquid chromatography. The time course of plosphorylation of threonine in both a and fJ subunits was similar to the time course of the generation of Ca2+/calmodulin-independence.Moreover, the time course of phosphorylation of one set of peptides, referred to as peptide 1/1', present in both a and ,3 subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Threonine was the only amino acid phosphorylated in peptide 1/1'. An additional peptide, referred to as peptide 2, was phosphorylated in the .8 subunit.The time course of phosphorylation of peptide 2, which also contained only phosphothreonine, did not parallel the time course of the generation of Ca2+/calmodulin-independence. It is likely that the phosphorylation of a threonine residue on peptide 1/1' is responsible for the generation of Ca2 /calmodulin-independence of Ca2+/calmodulin-dependent protein kinase IL.

Section: Discussionmentioning

confidence: 99%

Ca2+/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca2+/calmodulin-independence.

Lai¹,

Nairn²,

Gorelick³

et al. 1987

“…Cam-K substrates include the neuronal cytoskeletal proteins synapsin I and microtubule-associated protein 2 (MAP-2), as well as tyrosine hydroxylase, r, trytophan hydoxylase, and myelin basic protein (4). CaM-K enzymatic activities in nonneuronal tissues appear to be isozymes with different subunit size and composition (3,13).…”

mentioning

confidence: 99%

Molecular cloning of a brain-specific calcium/calmodulin-dependent protein kinase.

Lin¹,

Kapiloff²,

Durgerian³

et al. 1987

240

181

A calcium/calmodulin-dependent protein kinase type II (CaM-K) a-subunit cDNA has been cloned from rat brain. This enzyme is encoded by a 5.1-kilobase mRNA expressed exclusively in the brain. Hybridization histochemistry reveals that the CaM-K mRNA expression corresponds to the distribution of the immunoreactive a-subunit protein, suggesting that the high enzyme levels in specific brain areas reflect regional differences in gene expression. The sequence of CaM-K a-subunit cDNA indicates a 478-amino acid (54-kDa) protein with three functional domains. The domain organization suggests a structural model for calcium/calmodulindependent and independent states that might subserve shortand long-term responses to transient stimuli.

“…CaM Kinase II isoenzymes from skeletal muscle (28), liver (29,30), lung (31), and heart (32) have also been isolated, and the wide distribution of CaM kinase II immunoreactive proteins was demonstrated (33,34). Moreover, a/p-subunit DNA or RNA probes reacted with mRNA species ranging in size from 2.7 kb to 5.7 kb in various peripheral tissues and cultured cells (19)(20)(21)(22).…”

mentioning

confidence: 99%

Sequence analysis and DNA-protein interactions within the 5' flanking region of the Ca2+/calmodulin-dependent protein kinase II alpha-subunit gene.

Sunyer

Sahyoun

1990

The 5' flanking region of the brain Ca2+/ calmodulin-dependent protein kinase II a-subunit gene was identified and characterized. A total of 430 bases was sequenced upstream from the translation initiation codon, and the site of transcription initiation was located at -149 or -147 bases as determined by primer extension and S1 nuclease protection analysis, respectively. TATA and CAAT boxes were absent from their standard positions; however, the 5' flanking region was rich in G+C and contained a GGGCG and a TATATAA sequence 76 and 160 bases upstream from the transcription initiation site, respectively. Moreover, the sequence CAACGG was found 85 and 146 bases upstream from this site, indicating presumptive binding sites for the Myb protein. Gel-mobility shift assays revealed that a 120-base-pair fragment, which included the G+C-rich, TATA, and CAACGG sequences bound nuclear proteins specifically. DNA-protein complexes with similar gel mobilities were obtained with nuclear extracts from rat forebrain or cerebellum and from neonatal or adult brains. Extracts from rat liver, kidney, and spleen generated specific DNA-protein complexes with different electrophoretic mobilities, suggesting the occurrence of different nuclear proteins that bind to 5' regulatory elements of the Ca2+/calmodulin-dependent kinase II asubunit gene.Neuronal multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) represents a major Ca2+-operated molecular switch subject to regulation by Ca2+/ calmodulin and by autophosphorylation (1, 2). This abundant enzyme is located within different subcellular compartments including cytosol, cytoskeleton, nuclei, presynaptic terminals, and postsynaptic densities (3-13) and may regulate important neuronal functions such as neurotransmitter release and long-term potentiation (14-17).Soluble CaM kinase II was initially purified from brain as a 10-to 12-subunit oligomer with an apparent Mr of -600,000, composed primarily of two related a and P subunits. The a-to-,8 ratio varies from 3 or 4:1 in forebrain to 1:4 in cerebellum (3-5, 18). A third relatively minor subunit, p', has also been identified (3). a and 8 subunits are encoded by distinct genes (19-21), whereas ,3' is generated by alternative splicing of the 8-gene transcript (22).Enzyme levels and subunit composition were dependent on the stage of neuronal development; the levels increased dramatically between the second and fourth postnatal weeks in rat brain (23)(24)(25)(26)(27). The use of cDNA probes revealed that the a and 8 subunits of CaM kinase II are encoded by 5-to 5.4-kilobase (kb) and 3.9-to 4.8-kb mRNA species, respectively (19-21), and that the level of the 5-kb mRNA species increased 10-fold during the first 3 postnatal weeks in rat forebrain; a further 2.5-fold increase occurred by the end of 90 days (19).CaM Kinase II isoenzymes from skeletal muscle (28), liver (29, 30), lung (31), and heart (32) have also been isolated, and the wide distribution of CaM kinase II immunoreactive proteins was demonstrated (33, 34)...