Regular ArticleCa2+/Calmodulin-Dependent Protein Kinase V: Tissue Distribution and Immunohistochemical Localization in Rat Brain
Abstract
Polyclonal antibody against Ca2+/calmodulin-dependent protein kinase V (CaM kinase V) was prepared from guinea pigs immunized with synthetic polypeptide, based on the partial amino acid sequence of the rat brain enzyme. Immunoblot analysis of purified CaM kinase V revealed two immunoreactive bands with a molecular mass of 41 kDa and minor 40 kDa, respectively. Tissue distribution of CaM kinase V and immunohistochemical localization in rat brain were also investigated. Immunoblotting revealed the presence of immunoreactive proteins with the same molecular mass of 40 and 41 kDa, in the cerebrum, cerebellum, brain stem, pituitary gland, lung, adrenal gland, spleen, liver, colon, heart, stomach, ovary, spinal cord, and thymus. Immunohistochemistry revealed strong staining in the neuronal somata and weak staining in the nuclei. Densely stained regions included the cerebral cortex, the hippocampal formation, the caudatoputamen, the globus pallidus, the hypothalamus, the substantia nigra, the medial geniculate body, the olfactory bulb, the cerebellar cortex and the choroid plexus. CaM kinase V revealed different substrate specificity compared with CaM kinase II. These results lead to the notion that CaM kinase V may exist in 40- and 41-kDa isoforms, is widely distributed in various tissues, and may play an important role in the control of a wide variety of calcium regulated processes in the respective tissues.
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Phosphorylation screening identifies translational initiation factor 4GII as an intracellular target of Ca<sup>2+</sup>/calmodulin-dependent protein kinase I
2003, Journal of Biological ChemistryCaMKI is a Ca2+/calmodulin-dependent protein kinase that is widely expressed in eukaryotic cells and tissues but for which few, if any, physiological substrates are known. We screened a human lung cDNA expression library for potential CaMKI substrates by solid phase in situ phosphorylation (“phosphorylation screening”). Multiple overlapping partial length cDNAs encoding three proteins were detected. Two of these proteins are known: 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase and eukaryotic translation initiation factor (eIF) 4GII. To determine whether CaMKI substrates identified by phosphorylation screening represent authentic physiological targets, we examined the potential for [Ca2+]i- and CaMKI-dependent phosphorylation of eIF4GII in vitro and in vivo. Endogenous eIF4GII immunoprecipitated from HEK293T cells was phosphorylated by CaMKI, in vitro as was a recombinant fragment of eIF4GII encompassing the central and C-terminal regions. The latter phosphorylation occurred with favorable kinetics (Km = 1 μm; kcat = 1.8 s–1) at a single site, Ser1156, located in a segment of eIF4GII aligning with the phosphoregion of eIF4GI. Phosphopeptide mapping and back phosphorylation experiments revealed [Ca2+]i-dependent, CaMKI site-specific, eIF4GII phosphorylation in vivo. This phosphorylation was blocked by kinase-negative CaMKI consistent with a requirement for endogenous CaMKI for in vivo eIF4GII phosphorylation. We conclude that phosphorylation screening is an effective method for searching for intracellular targets of CaMKI and may have identified a new role of Ca2+ signaling to the translation apparatus.
Components of a calmodulin-dependent protein kinase cascade: Molecular cloning, functional characterization and cellular localization of Ca<sup>2+</sup>/calmodulin-dependent protein kinase kinase β
1999, Journal of Biological ChemistryCa2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain VIII for maximal activity. Two distinct CaMKI/IV kinases, CaMKKα and CaMKKβ, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996)J. Biol. Chem. 271, 10806–10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKKβ, tissue and regional brain localization of CaMKKβ protein, and mRNA and functional characterization of recombinant CaMKKβ in vitro and in Jurkat T cells. The sequences of human and rat CaMKKβ demonstrate 65% identity and 80% similarity with CaMKKα and 30–40% identity with CaMKI and CaMKIV themselves. CaMKKβ is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKKβ tracks more closely with CaMKIV than does CaMKKα. Bacterially expressed CaMKKβ undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKKβ activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKKβ activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.
Unique inhibitory action of the synthetic compound 2-[N-(2-aminoethyl)-N-(5-isoquinolinesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (CKA-1306) against calcium/calmodulin-dependent protein kinase I
1998, Biochemical PharmacologyA newly synthesized compound, 2-[N-(2-aminoethyl)-N-(5-isoquinolinesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (CKA-1306), was found to inhibit cyclic AMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase I (CaMK I) with ic50 values of 1.6 ± 0.14 and 2.5 ± 0.16 μM, respectively. In contrast, the established PKA inhibitors H-8 and H-89 inhibited CaMK I with relatively high ic50 values of >100 and 24.4 ± 3.2 μM, respectively. An additional inhibitor, KN-62, against Ca2+/calmodulin-dependent protein kinase II (CaMK II) did not inhibit either PKA or CaMK I at the concentrations tested. In our library of many isoquinolinesulfonamide derivatives, only CKA-1306 inhibited CaMK I to a satisfactory degree, suggesting a unique mode of action. Indeed, the inhibition of CaMK I by CKA-1306 was competitive in every respect to Mg2+/ATP, peptide substrate (syntide-2), and Ca2+/calmodulin. This phenomenon may be understood from the context of the recently determined structure of the enzyme in its autoinhibited state. Such kinetic analysis was also extended to cases using a phosphorylated and activated enzyme at Thr177 or a constitutively active, COOH-terminal truncated mutant at Gln293. CKA-1306 still competed with Mg2+/ATP for the two enzymes, but it no longer achieved any competitive advantage over syntide-2. These results may reflect some differences in the active conformation of CaMK I. However, the compound should be constant in its recognition of an Mg2+/ATP-binding site of the enzyme. Though CKA-1306 is not specific to CaMK I, the compound will be useful in studying the enzyme further under limited conditions.
While changes in gene expression are critical for many brain functions, including long-term memory, little is known about the cellular processes that mediate stimulus–transcription coupling at central synapses. In studying the signaling pathways by which synaptic inputs control the phosphorylation state of cyclic AMP–responsive element binding protein (CREB) and determine expression of CRE-regulated genes, we found two important Ca2+/calmodulin (CaM)–regulated mechanisms in hippocampal neurons: a CaM kinase cascade involving nuclear CaMKIV and a calcineurin-dependent regulation of nuclear protein phosphatase 1 activity. Prolongation of the synaptic input on the time scale of minutes, in part by an activity-induced inactivation of calcineurin, greatly extends the period over which phospho-CREB levels are elevated, thus affecting induction of downstream genes.
Identification and purification of Ca<sup>2+</sup>/calmodulin-dependent protein kinase V from human gastric carcinoma
1996, Biochimica et Biophysica Acta - Molecular Basis of DiseaseWe previously purified a novel Ca2+/calmodulin-dependent protein kinase (CaM kinase) V, which has proven to be a member of the CaM kinase I family. Immunohistochemical staining of surgically-resected specimens from human subjects using specific antibody which reacts with CaM kinases I and V demonstrated heterogeneous distribution of CaM kinase I/V in normal gastric mucosa. The kinase was located mainly at the bottom of foveoral epithelium and in the gastric gland (< 25% immunopositive). In contrast, this kinase was abundant in various types of gastric carcinomas (> 75%), but not in gastric adenomas. Preferential and consistent presence of this kinase was confirmed by immunoblot analysis of gastric carcinoma and human gastric cancer cell lines, Kato-III and MKN-45. CaM kinase I/V was co-purified with CaM kinase II from resected gastric carcinoma using anion-exchange chromatography followed by calmodulin-affinity chromatography. The two kinases were finally separated by HPLC-based gel filtration. Purified CaM kinase I/V from gastric carcinoma did not possess detectable autophosphorylating activity, in contrast to CaM kinase II. The findings suggest CaM kinase I/V may possess abnormal biochemical properties in human gastric carcinoma, and the kinase could participate in cell growth of the carcinoma.
Structure, Regulation, and Function of Calcium/Calmodulin-Dependent Protein Kinase I
1996, Advances in PharmacologyIn particular, a large number of the effects of Ca2+/CaM are mediated through the regulation of protein phosphorylation and dephosphorylation. A family of Ca2+/CaM-dependent protein kinases (CaM kinases) has been identified. The first two members identified, myosin light-chain kinase (MLCK) and phosphorylase kinase, were found to be highly specific for their respective substrates, myosin P light chain and phosphorylase. This led to the initial belief that the CaM kinases were monofunctional kinases that were distinct from the multifunctional protein kinases such as CAMP-dependent protein kinase (PKA) or protein kinase C (PKC). The CaM kinases share many common features. They each have a highly conserved catalytic domain and a COOH-terminal regulatory domain that is not as highly conserved among family members. In addition, several of the enzymes have NH2- and/or COOH-terminal extensions that are involved in interaction with other subunits or in targeting to specific subcellular locations. Irrespective of the exact relationship of CaM kinase W and CaM kinases Ia and Ib, CaM kinase I is the smallest member of the CaM kinase family. Based on extensive biochemical and structural analyses of the CaM kinases, as well as other second messenger-regulated protein kinases, it has been proposed that this class of enzyme is regulated by an intrasteric autoinhibitory mechanism. The central feature of this mechanism is that amino acid residues within the regulatory domain bind to the active site of the kinase and sterically block the access of peptide substrate and/ or MgATP.