Functional analysis of a complementary DNA for the 50-kilodalton subunit of calmodulin kinase II.

The calcium-calmodulin-dependent protein kinase II is a major component of brain synaptic junctions and has been proposed to play a variety of important roles in brain function. A complementary DNA representing a portion of the smaller 50-kilodalton subunit of the rat brain enzyme has been cloned and sequenced. The calmodulin-binding region has been identified and a synthetic analog prepared that binds calmodulin with high affinity in the presence of calcium. Like the 50-kilodalton kinase polypeptide, the concentration of the messenger RNA varies both neuroanatomically and during postnatal development of the brain. The broad tissue and species cross-reactivity of the complementary DNA suggests that the 50-kilodalton subunit found in rat brain is evolutionarily conserved and is the product of a single gene.

Cytochrome c mRNA levels decrease in senescent rat heart.

The concentration of mitochondria decreases in the heart as rodents age from maturity to senescence. The reason for this change is not known. One purpose of the present study was to determine if cytochrome c mRNA, representative of proteins of the inner mitochondrial membrane, decreased in the hearts of Fischer 344 rats as they aged from 12 to 24 months. Twenty-two percent less cytochrome c mRNA existed per given quantity of extracted RNA from the heart in 24-month-old rats as compared with the 12-month-old group. No change in the quantities of cardiac alpha-actin mRNA, Ca2+/calmodulin protein kinase II mRNA or 18S rRNA was noted between 12- and 24-month-old hearts. Thus, the decrease in cytochrome c mRNA suggests that decreases in mRNAs for proteins of the inner mitochondrial membrane could play some role in the diminished concentration of mitochondria that exists in the senescent heart.

Increase in liver protein phosphatase-1 in spontaneously diabetic Chinese hamsters.

Two broad-specifically protein phosphatases, termed protein phosphatase-1 (PrP-1) and protein phosphatase-2A (PrP-2A), accounting for all the hepatic activity regulating glycogen phosphorylase, were measured in spontaneously diabetic Chinese hamsters exhibiting persistent glycosuria. When compared with genetically related inbred sublines free of glycosuria, diabetic animals demonstrated approximately 25% increase in PrP-1 activity measured either in crude tissue extracts or in cytosols fractionated by ion-exchange chromatography. No significant alteration in total PrP-2A activity was observed in the diabetic animals. These findings indicate that a specific change in hepatic PrP-1 is associated with genetically acquired diabetes in Chinese hamsters. In contrast to reported data using animals with experimentally induced diabetes mellitus, hepatic PrP-1 was increased in the spontaneously diabetic Chinese hamsters. The data suggests that distinct alterations in PrP-1 and associated metabolic consequences are exhibited by different types of diabetes.

'Diabetic' emergencies. They happen with or without diabetes.

Ketoacidosis, severe hyperosmolality due to hyperglycemia, and severe hypoglycemia are all life-threatening emergencies that often occur in the absence of any history of diabetes mellitus. The key to management of diabetic ketoacidosis is understanding that treatment is aimed more at the breakdown and metabolism of triglycerides in adipose tissue than at hyperglycemia per se. The diabetic hyperosmolar state is most easily treated with aggressive fluid management, with the caveat that too-rapid administration of hypotonic fluids may increase the already significant mortality from this condition. Life-threatening hypoglycemia most commonly occurs with administration of oral hypoglycemic drugs or insulin, although other drugs and any malnourished state may also be precipitating factors. Acute administration of glucagon or dextrose alleviates life-threatening hypoglycemia. Success in managing these diabetic emergencies depends on rapidity of recognition and institution of direct treatment measures.

The second-messenger system for peptide hormones.

There are three major hormone classes--peptide, steroid, and the newly defined growth factors--each with its own system for signal transduction in the cell. Two interdependent theses are proposed for the peptide hormone: that incoming signal transduction requires coupling to a G protein in a second-messenger pathway, and that second-messenger redundancy assures checks and balances in cell regulation.

The effect of phorbol esters on the expression of mRNA for the alpha subunit of calcium/calmodulin dependent protein kinase II in rabbit renal proximal tubules.

Calcium-calmodulin dependent protein kinase II (CaM-KII) has been implicated in the inhibition of Na(+)-H+ exchange activity in the brush border of the renal proximal convoluted tubule. Conversely, the activity of the antiporter is stimulated in response to phosphorylation by calcium-phospholipid dependent protein kinase (PKC). In these experiments, we explored the potential for direct interaction between these two protein kinases by determining the effect of PKC activation by tumor promoting phorbol esters on the expression of mRNA for CaM-KII in the rabbit renal proximal tubule. The results indicate that activation of PKC reduced the steady-state levels of the mRNA for the alpha subunit of CaM-KII in a dose and time dependent manner. This suggests a novel mechanism by which PKC can antagonize the action of CaM-KII in selected tissues.

Identification of high-affinity calmodulin-binding proteins in rat liver.

The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or "acceptor" proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised greater than 80% of the Ca2+-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding "subunit" of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.

Identification of calcium-calmodulin multifunctional protein kinase II in rabbit kidney.

Recent studies have demonstrated that calcium/calmodulin-dependent multifunctional protein kinase II (CaM-KII) inhibits the reconstituted Na(+)-H+ exchanger from the brush border membrane of proximal convoluted tubule of the rabbit kidney. The present studies were undertaken to evaluate the physiological relevance of this finding by establishing the presence of CaM-KII in rabbit kidney and proximal convoluted tubule cells by Northern RNA hybridization analysis to demonstrate the messenger RNA (mRNA) for CaM-KII and by a selective enzymatic assay of CaM-KII using a synthetic peptide substrate. A single 4.9 Kb mRNA was observed on hybridization of total RNA from rabbit kidney cortex and medulla and from an enriched suspension of rabbit kidney proximal convoluted tubules with a cDNA for rat brain CaM-KII. An enzyme assay using a synthetic peptide substrate representing the site phosphorylated by CaM-KII on glycogen synthase demonstrated calcium-calmodulin dependent protein kinase activity in both rabbit kidney cortex (specific activity of 662 +/- 127 nmol.min-1.mg protein-1) and proximal tubule cells (546 +/- 77 nmol.min-1.mg protein-1). These data establish the presence of CaM-KII in the rabbit kidney, and suggest a role for this enzyme in the control of renal electrolyte transport.

Mapping of calmodulin-binding domain of Ca2+/calmodulin-dependent protein kinase II from rat brain.

Recent molecular cloning experiments have identified a 25 amino-acid region as the calmodulin-binding domain of the alpha-subunit of rat brain Ca2+/calmodulin-dependent multifunctional protein kinase II (CaM-K II). Synthetic peptides, derived from the deduced amino-acid sequence encompassing this region, were examined for their ability to bind calmodulin in a calcium dependent manner and to inhibit the Ca2+/calmodulin-dependent autophosphorylation of CaM-K II. Comparison of these structure-function relationships highlighted a region of 5 amino-acids, which was essential for calmodulin interaction and inhibition of kinase activity. This region demonstrated some homology with other calmodulin-binding peptides, and may represent a key site of interaction of the kinase with calmodulin. These analyses provide additional insight into the molecular mechanism underlying the Ca2+ regulation of CaM-K II.

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues.

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.

Chimeric calmodulin-cardiac troponin C proteins differentially activate calmodulin target enzymes.

To evaluate the role of domain I of calmodulin (CaM) in the activation of target enzymes, a series of CaM mutants was constructed in which domain I (49 amino acids) was substantially deleted, or was exchanged with the homologous region (58 amino acids) of cardiac troponin C (cTnC). The proteins are 1) aM, a mutant CaM in which domain I has been deleted; 2) TaM, first domain of cTnC, last three domains of CaM; 3) TaM-BMI, same as TaM, except the nonfunctional first Ca2(+)-binding domain has been restored by mutagenesis; 4) CaT, first domain of CaM, last three domains of cTnC. These proteins were evaluated for Ca2+ binding properties and as activators of three CaM target enzymes, CaM-dependent phosphodiesterase (PDE), smooth muscle myosin light chain kinase (MLCK), and CaM-dependent multifunctional protein kinase (CaM kinase II). The chimeric proteins containing four domains bound Ca2+ in the manner expected from the number and nature of EF hands. In contrast, aM bound only two Ca2+, suggesting that deletion of domain I may have disrupted binding in one of the remaining three domains, and did not activate the three enzymes. The kinetics of activation of PDE by CaM, TaM, and TaM-BMI were identical. Although cTnC and CaT could maximally activate PDE, the Kact for these mutants were greater than 2000 times than for CaM. All mutated proteins except CaT were poor activators of CaM kinase II and this protein activated the kinase to 65% that of CaM, with a nearly identical Kact. CaT and TaM, were poor agonists of MLCK. Activation of Ca2(+)-binding site I in TaM (TaM-BMI), completely prevented activation of MLCK. In addition, TaM-BMI was a potent competitive inhibitor of MLCK activation by CaM (Ki = 66 nM). We conclude 1) a domain I is necessary to activate these target enzymes, and the substitution of the corresponding region of cTnC into CaM leads to differential effects; 2) an active first Ca2(+)-binding site is not essential for activation of PDE and the primary sequence of the first domain of CaM need not be highly conserved; 3) for CaM kinase II, determinants in the first domain are critical whereas more flexibility exists for the remaining three domains; 4) since TaM-BMI acts as a potent competitive inhibitor of MLCK binding of CaM to a target enzyme and activation can be dissociable events.