scholarly journals Molecular and biochemical characterization of a calcium/calmodulin-binding protein kinase from rice

2002 ◽  
Vol 368 (1) ◽  
pp. 145-157 ◽  
Author(s):  
Lei ZHANG ◽  
Bi-Feng LIU ◽  
Shuping LIANG ◽  
Russell L. JONES ◽  
Ying-Tang LU
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Binding Protein ◽  
Enzymic Activity ◽  
Dependent Manner ◽  
Surface Plasmon Resonance Analysis ◽  
Calmodulin Binding ◽  
Dependent Protein

A Ca2+/calmodulin (CaM)-binding protein kinase from rice (Oryza sativa), OsCBK, has been characterized that lacks Ca2+-binding EF hands and has Ca2+/CaM-independent autophosphorylation and substrate-phosphorylation activity. OsCBK has all 11 subdomains of a kinase catalytic domain and a putative CaM-binding domain, and shares high identity with Ca2+-dependent-protein-kinase ('CDPK')-related protein kinases in plants. OsCBK bound CaM in a Ca2+-dependent manner as previously reported for Ca2+/calmodulin-dependent protein kinases in animals, but autophosphorylation and phosphorylation of histone IIIs were Ca2+/CaM-independent. Surface plasmon resonance analysis showed that OsCBK specifically bound CaM with high affinity (KD = 30nM). Capillary electrophoresis showed that phosphorylation of OsCBK occurred on serine and threonine residues. These data show that OsCBK is a serine/threonine protein kinase that binds Ca2+/CaM, but whose enzymic activity is independent of Ca2+/CaM. In situ hybridization showed that OsCBK is expressed in reproductive and vegetative tissues of rice and shows temporal and spatial changes during plant growth and development. OsCBK is highly expressed in zones of cell division and it is particularly abundant in sporogenous cells of the anther at meiosis.

scholarly journals A tobacco (Nicotiana tabaccum) calmodulin-binding protein kinase, NtCBK2, is regulated differentially by calmodulin isoforms

2003 ◽  
Vol 376 (1) ◽  
pp. 291-302 ◽  
Author(s):  
Wei HUA ◽  
Shuping LIANG ◽  
Ying-Tang LU
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Dissociation Constants ◽  
Binding Protein ◽  
Enzymic Activity ◽  
Protein Motif ◽  
Independent Manner ◽  
Nicotiana Tabaccum ◽  
Calmodulin Binding ◽  
Dependent Protein

A calcium (Ca2+)/calmodulin (CaM)-binding protein kinase (CBK) from tobacco (Nicotiana tabaccum), NtCBK2, has been characterized molecularly and biochemically. NtCBK2 has all 11 conserved subdomains of the kinase-catalytic domain and a CaM-binding site as shown by other kinases, including Ca2+-dependent protein kinase and chimaeric Ca2+/CaM-dependent protein kinases. However, this kinase does not contain an EF-hand motif for Ca2+ binding, and its activity was not regulated by Ca2+. Whereas NtCBK2 phosphorylated both itself and other substrates, such as histone IIIS and syntide-2, in a Ca2+/CaM-independent manner, as also shown by OsCBK, a CaM-binding protein kinase from rice (Oryza sativa), the kinase activity of NtCBK2 was greatly stimulated by Ca2+/CaM, whereas that of OsCBK was not. By molecular dissection analyses, the CaM-binding domain of NtCBK2 has been localized in a stretch of 30 amino acid residues at residue positions 431–460 as a 1-5-10 protein motif. Three tobacco CaM isoforms (NtCaM1, NtCaM3 and NtCaM13) used in the present study have been shown to bind to NtCBK2, but with different dissociation constants (Kds), as follows: NtCaM1, 55.7 nM; NtCaM3, 25.4 nM; and NtCaM13, 19.8 nM, indicating that NtCBK2 has a higher affinity for NtCaM3 and NtCaM13 than for NtCaM1. The enzymic activity of NtCBK2 was also modulated differently by various CaM isoforms. Whereas the phosphorylation activity of NtCBK2 was shown by assay to be enhanced only ≈2–3-fold by the presence of NtCaM1, the activity could be amplified up to 8–9-fold by NtCaM3 or 10–11-fold by NtCaM13, suggesting that NtCaM3 and NtCaM13 are better activators than NtCaM1 for NtCBK2.

scholarly journals Characterization of a calmodulin-regulated Ca2+-dependent-protein-kinase-related protein kinase, AtCRK1, from Arabidopsis

2004 ◽  
Vol 383 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Ying WANG ◽  
Shuping LIANG ◽  
Qi-Guang XIE ◽  
Ying-Tang LU
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Related Protein ◽  
Dependent Protein Kinase ◽  
Independent Manner ◽  
Calmodulin Binding ◽  
Kinase Catalytic Domain ◽  
Dependent Protein

An AtCRK1 [Arabidopsis thaliana CDPK (Ca2+-dependent protein kinase)-related protein kinase 1] has been characterized molecularly and biochemically. AtCRK1 contains the kinase catalytic domain and a CaM (calmodulin)-binding site. Our results demonstrated that AtCRK1 could bind CaM in a Ca2+-dependent manner. This kinase phosphorylated itself and substrates such as histone IIIS and syntide-2 in a Ca2+-independent manner and the activity was stimulated by several CaM isoforms through its CaM-binding domain. This domain was localized within a stretch of 39 amino acid residues at positions from 403 to 441 with Kd=67 nM for CaM binding. However, the stimulation amplification of the kinase activity of AtCRK1 by different CaM isoforms was similar.

scholarly journals Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+ -dependent protein kinases

1982 ◽  
Vol 202 (1) ◽  
pp. 217-224 ◽  
Author(s):  
N Katoh ◽  
R L Raynor ◽  
B C Wise ◽  
R C Schatzman ◽  
R S Turner ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Dependent Protein Kinase ◽  
Calmodulin Binding ◽  
Substrate Protein ◽  
Dependent Protein ◽  
A Site

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.

scholarly journals A novel Ca2+/calmodulin-dependent protein kinase and a male germ cell-specific calmodulin-binding protein are derived from the same gene.

1991 ◽  
Vol 11 (8) ◽  
pp. 3960-3971 ◽  
Author(s):  
A R Means ◽  
F Cruzalegui ◽  
B LeMagueresse ◽  
D S Needleman ◽  
G R Slaughter ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Germ Cell ◽  
Binding Protein ◽  
Single Gene ◽  
Transcription Unit ◽  
Male Germ Cell ◽  
Dependent Protein Kinase ◽  
Calmodulin Binding ◽  
Brain Cdna Library ◽  
Dependent Protein

A cDNA representing a unique Ca2+/calmodulin-dependent protein kinase has been cloned and sequenced from a rat brain cDNA library. This enzyme, expressed in brain, testis, and spleen, is only 32% identical to the various isoforms of Ca2+/calmodulin-dependent protein kinase II. The sequence of the COOH-terminal 169 amino acids is identical to that of a previously described male germ cell-specific calmodulin-binding protein called calspermin (T. Ono, G.R. Slaughter, R.G. Cook, and A.R. Means, J. Biol. Chem. 264:2081-2087, 1989). This identity extends to the nucleic acid sequence and includes all but the first 130 nucleotides of the calspermin cDNA. Primer extension and sequence of a genomic fragment containing the unique calspermin sequence reveals that this mRNA is derived from the kinase transcription unit by germ cell-specific use of a unique exon. In situ hybridization was used to demonstrate that both kinase and calspermin mRNAs are expressed during spermatogenesis. The kinase mRNA is first detected in early meiotic cells and declines to a low level in haploid cells. Calspermin mRNA first appears in pachytene primary spermatocytes and continues to increase as cells complete meiosis and undergo terminal differentiation. These results show that differential utilization of a single gene during spermatogenesis is used to generate mRNAs that encode proteins with distinct functions.

scholarly journals Phosphorylation-dependent subcellular translocation of a Ca2+/calmodulin-dependent protein kinase produces an autonomous enzyme in Aplysia neurons.

1985 ◽  
Vol 100 (3) ◽  
pp. 835-842 ◽  
Author(s):  
T Saitoh ◽  
J H Schwartz
Keyword(s):  
Protein Kinase ◽  
Subcellular Distribution ◽  
Kinase Activity ◽  
Binding Protein ◽  
Second Messengers ◽  
Dependent Protein Kinase ◽  
Calmodulin Binding ◽  
Aplysia Neurons ◽  
Membrane Cytoskeleton ◽  
Dependent Protein

We have shown previously that the subcellular distribution of a major calmodulin-binding protein is altered under conditions causing increased synthesis of cAMP in Aplysia neurons (Saitoh, T., and J. H. Schwartz, 1983, Proc. Natl. Acad. Sci. USA, 80:6708-6712). We now provide evidence that this Mr 55,000 protein is a subunit of a Ca2+/calmodulin-dependent kinase: (a) both the Mr 55,000 calmodulin-binding protein and kinase activity are loosely attached to the membrane-cytoskeletal complex; (b) both kinase activity and the Mr 55,000 protein are translocated from the membrane-cytoskeleton complex to the cytoplasm under conditions that cause the change in the subcellular distribution of the Mr 55,000 calmodulin-binding protein; and (c) calmodulin-binding activity of the Mr 55,000 protein and the ability to carry out the Ca2+/calmodulin-dependent phosphorylation of synapsin I are purified in parallel. The subcellular localization of the Ca2+/calmodulin-dependent protein kinase appears to be under control of two second messengers: Ca2+ and cAMP. We find that the Mr 55,000 subunit is phosphorylated when the extracted membrane-cytoskeleton complex is incubated with Ca2+, calmodulin, and ATP, with the concomitant release of this phosphorylated peptide from the complex. Previously, we had found that, when translocation occurs in extracts in the presence of cAMP and ATP (but in the absence of Ca2+), there was no detectable phosphorylation of the Mr 55,000 subunit itself. The subcellular distribution of the subunit thus appears to be influenced by (a) cAMP-dependent phosphorylation, which, we infer, modifies some as yet unidentified structural component, causing the release of the enzyme; and (b) Ca2+/calmodulin-dependent phosphorylation of the Mr 55,000 subunit. These studies also suggest that phosphorylation has an important regulatory consequence: during the Ca2+/calmodulin-dependent translocation of the Mr 55,000 subunit, the kinase appears to be activated, becoming independent of added Ca2+/calmodulin.

scholarly journals Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

1990 ◽  
Vol 1 (13) ◽  
pp. 1015-1025 ◽  
Author(s):  
M V Autieri ◽  
K L Fresa ◽  
F D Coffman ◽  
M E Katz ◽  
S Cohen
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Protein Kinases ◽  
Phorbol Esters ◽  
Adenosine Monophosphate ◽  
Peripheral Blood Lymphocytes ◽  
Lymphoid Cells ◽  
Dependent Manner ◽  
Dependent Protein

We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein.

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