A signaling complex of Ca2+-calmodulin-dependent protein kinase IV and protein phosphatase 2A.

Stimulation of T lymphocytes results in a rapid increase in intracellular calcium concentration ([Ca2+]i) that parallels the activation of Ca2+-calmodulin-dependent protein kinase IV (CaMKIV), a nuclear enzyme that can phosphorylate and activate the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). However, inactivation of CaMKIV occurs despite the sustained increase in [Ca2+]i that is required for T cell activation. A stable and stoichiometric complex of CaMKIV with protein serine-threonine phosphatase 2A (PP2A) was identified in which PP2A dephosphorylates CaMKIV and functions as a negative regulator of CaMKIV signaling.

The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1.

The cis/trans peptidyl-prolyl isomerase, Pin1, is a regulator of mitosis that is well conserved from yeast to man. Here we demonstrate that depletion of Pin1-binding proteins from Xenopus egg extracts results in hyperphosphorylation and inactivation of the key mitotic regulator, Cdc2/cyclin B. We show biochemically that this phenotype is a consequence of Pin1 interaction with critical upstream regulators of Cdc2/cyclin B, including the Cdc2-directed phosphatase, Cdc25, and its known regulator, Plx1.

Characterization of substrate phosphorylation and use of calmodulin mutants to address implications from the enzyme crystal structure of calmodulin-dependent protein kinase I.

Calcium/calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to the enzyme and indirectly promotes the phosphorylation and synergistic activation of CaMKI by an exogenous kinase. We have evaluated the initial CaM-dependent activation of the unphosphorylated form of CaMKI. The kinetics of bacterially expressed human CaMKI show that the peptide syntide-2 is a relatively poor substrate, whereas the synapsin site-1 peptide is 17-fold more specific.

Defective survival and activation of thymocytes in transgenic mice expressing a catalytically inactive form of Ca2+/calmodulin-dependent protein kinase IV.

We have generated transgenic mice that express a catalytically inactive form of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) specifically in thymic T cells. The presence of this protein results in a markedly reduced thymic cellularity, although the distribution of the remaining cells is normal based on evaluation of the CD4 and CD8 cell surface antigens that are used to gauge T cell development. Isolated thymic T cells from the transgenic mice also show a dramatically decreased survival rate when evaluated in culture under conditions that do not favor activation.

The structure of a calmodulin mutant with a deletion in the central helix: implications for molecular recognition and protein binding.

Background: Calmodulin (CaM) is the major calcium-dependent regulator of a large variety of important intracellular processes in eukaryotes. The structure of CaM consists of two globular calcium-binding domains joined by a central 28-residue alpha helix. This linker helix has been hypothesized to act as a flexible tether and is crucial for the binding and activation of numerous target proteins.

The CRABP I gene contains two separable, redundant regulatory regions active in neural tissues in transgenic mouse embryos.

The CRABP I gene is expressed in a spatiotemporal pattern in neural and mesenchymal tissues at the onset of organogenesis. The neural pattern of CRABP I expression includes specific rhombomeres of the hindbrain, neural crest cells and their derivatives the optic stalk, and the central area of the neural retina. We have created transgenic mouse lines with CRABP I 5' and transcribed regions fused to the lacZ structural gene that recapitulate much of this neural pattern of expression.

Functional consequences of truncating amino acid side chains located at a calmodulin-peptide interface.

To test the relevance of the calmodulin-peptide crystal structures to their respective calmodulin-enzyme interactions, amino acid side chains in calmodulin were altered at positions that interact with the calmodulin-binding peptide of smooth muscle myosin light chain kinase but not with the calmodulin kinase IIalpha peptide. Since shortening the side chains of Trp-800, Arg-812, and Leu-813 in smooth muscle myosin light chain kinase abrogated calmodulin-dependent activation (Bagchi, I. C.

Expression of a constitutively active Ca2+/calmodulin-dependent kinase in Aspergillus nidulans spores prevents germination and entry into the cell cycle.

The unique gene for Ca2+/calmodulin-dependent protein kinase (CaMK) has been shown to be essential in Aspergillus nidulans. Disruption of the gene prevents entry of spores into the nuclear division cycle. Here we show that expression of a constitutively active form of CaMK also prevents spores from entering the first S phase in response to a germinating stimulus.

Regulation and properties of the rat Ca2+/calmodulin-dependent protein kinase IV gene and its protein products.

Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric multifunctional enzyme that is expressed only in subanatomical portions of the brain, T lymphocytes, and postmeiotic male germ cells. It is present in the nucleus of the cells in which it is expressed and can phosphorylate and activate the cyclic AMP response element binding proteins CREB and CREM tau in a manner analogous to protein kinase A. In the absence of Ca2+/calmodulin, CaMKIV is inactive.

Methionine to glutamine substitutions in the C-terminal domain of calmodulin impair the activation of three protein kinases.

The 9 methionine residues of vertebrate calmodulin (CaM) were individually changed to glutamine residues in order to investigate their roles in enzyme binding and activation. The mutant proteins showed three classes of effect on the activation of smooth muscle myosin light chain kinase, CaM-dependent protein kinase IIalpha, and CaM-dependent protein kinase IV. First, some mutations had no appreciable effect on the ability of CaM to activate the three protein kinases.

cAMP-response element modulator tau is a positive regulator of testis angiotensin converting enzyme transcription.

Testis angiotensin-converting enzyme (ACE) is a unique form of ACE, only produced by male germ cells, and results from a testis-specific promoter found within the ACE gene. We have investigated the role of cAMP-response element modulator (CREM)tau in testis ACE transcription. In gel shift experiments, testes nuclear proteins retard an oligonucleotide containing the cAMP-response element (CRE) found at position -55 in the testis ACE promoter.

Ca(2+)/calmodulin-dependent kinase is essential for both growth and nuclear division in Aspergillus nidulans.

The calmodulin gene has been shown to be essential for cell cycle progression in a number of eukaryotic organisms. In vertebrates and Aspergillus nidulans the calmodulin dependence also requires calcium. We demonstrate that the unique gene encoding a multifunctional calcium/calmodulin-dependent protein kinase (CaMK) is also essential in A.

A unique phosphorylation-dependent mechanism for the activation of Ca2+/calmodulin-dependent protein kinase type IV/GR.

The activity of the Ca2+/calmodulin-dependent protein kinase IV/Gr (CaMKIV/Gr) is shown to be strictly regulated by phosphorylation of three residues both in vitro and in response to antigen receptor-mediated signaling in lymphocytes. One residue, Thr-200, is indispensable for enhancement of Ca2+/calmodulin-dependent basal activity by CaMKIV/Gr kinase. This event requires Ca2+/calmodulin in the full-length CaMKIV/Gr but is Ca2+/calmodulin-independent when a truncated version of CaMKIV/Gr is used as a substrate (DeltaCaMKIV/Gr1-317 (Delta1-317)).

FGF-2, BMP-2, and BMP-4 regulate retinoid binding proteins and receptors in 3T3 cells.

An Important part of intercellular signalling is the ability of responding cells to regulate multiple signal transduction pathways. Just as retinoic acid exposure alters expression of many peptide growth factors and their receptors, we have found that peptide growth factors alter the expression of cellular retinoic acid binding proteins (CRABPs) and retinoic acid receptors (RARs). FGF-2 (basic fibroblast growth factor) treatment of BALB 3T3 fibroblasts increased the level of CRABP I RNA, whereas bone morphogenetic protein (BMP)-2 and BMP-4 reduced this level as well as the levels of CRABP II and RAR beta 1/beta 3 transcripts.

Alterations in calcium channel currents underlie defective insulin secretion in a transgenic mouse.

A transgenic mouse overexpressing a mutant form of calmodulin (CaM-8) that is selectively targeted to pancreatic beta-cells has an impaired ability to secrete insulin in response to elevated blood glucose. Fluorescence measurements of cytosolic Ca2+ concentration ([Ca2+]i) showed that intracellular Ca2+ rises produced by glucose were smaller than normal in beta-cells of CaM-8 mice. Glucose utilization rates were not different between the CaM-8 and control beta-cells, suggesting that glucose metabolism was unperturbed by CaM-8.

HER-2/c-erbB2 is phosphorylated by calmodulin-dependent protein kinase II on a single site in the cytoplasmic tail at threonine-1172.

Calmodulin-dependent protein kinase II (Cam kinase II) is known to desensitise epidermal growth factor receptor (HER-1) tyrosine kinase activity by a process involving phosphorylation at serines 1046/47 in the cytoplasmic tail. We have developed an experimental system to investigate phosphorylation of the related HER-2/c-erbB2 proto-oncogene utilising purified Cam kinase II and recombinant glutathione-S-transferase (GST) fusion proteins. The cDNA for rat Cam kinase II-alpha was transfected into human embryonic kidney (HEK) 293 fibroblasts and the expressed protein purified to homogeneity by calmodulin-agarose affinity chromatography.

Multiple Ca(2+)-calmodulin-dependent protein kinase kinases from rat brain. Purification, regulation by Ca(2+)-calmodulin, and partial amino acid sequence.

We have purified to near homogeneity from rat brain two Ca(2+)-calmodulin-dependent protein kinase I (CaM kinase I) activating kinases, termed here CaM kinase I kinase-alpha and CaM kinase I kinase-beta (CaMKIK alpha and CaMKIK beta, respectively). Both CaMKIK alpha and CaMKIK beta are also capable of activating CaM kinase IV. Activation of CaM kinase I and CaM kinase IV occurs via phosphorylation of an equivalent Thr residue within the "activation loop" region of both kinases, Thr-177 and Thr-196, respectively.

Role of Ca++/calmodulin binding proteins in Aspergillus nidulans cell cycle regulation.

The goal of this review is to summarise the current knowledge concerning the targets of Ca++/calmodulin that are essential for cell cycle progression in lower eukaryotes. Emphasis is placed on Aspergillus nidulans since this is the only organism to date shown to posses essential Ca++ dependent calmodulin activated enzymes. Two such enzymes are the calmodulin activated protein phosphatase, calcineurin and the calmodulin dependent protein kinase.

Organization and analysis of the complete rat calmodulin-dependent protein kinase IV gene.

A 42-kilobase pair region of rat DNA containing the Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV) gene has been cloned and characterized. The gene consists of 12 exons and 11 introns and is predicted to encode both beta and alpha forms of CaM kinase IV as well as the testis-specific calmodulin-binding protein calspermin. The promoter utilized to generate the alpha-kinase isoform is located in intron 1, whereas the promoter utilized to produce the calspermin transcript is contained in intron 10.

Defective glycolysis and calcium signaling underlie impaired insulin secretion in a transgenic mouse.

Pancreatic beta cells from mice that overexpress the Ca(2+)-binding protein calmodulin have a unique secretory defect that leads to chronic hyperglycemia. To further understand the molecular basis underlying this defect, we have studied signaling pathways in these beta cells. Measurements of cytosolic free Ca2+ concentration ([Ca2+]i) using fura-2 or indo-1 revealed a markedly reduced response when glucose was the stimulant.

An intron facilitates activation of the calspermin gene by the testis-specific transcription factor CREM tau.

Calspermin is a high affinity Ca2+/calmodulin binding protein that is found only in postmeiotic male germ cells. Our previous studies have shown that the calspermin transcript is produced by utilization of a testis-specific promoter located within an intron of the calmodulin kinase IV gene. This promoter contains two cAMP response element-like motifs that bind the testis-specific transcription factor CREM tau.

Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase.

Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3.

Phosphorylation and activation of Ca(2+)-calmodulin-dependent protein kinase IV by Ca(2+)-calmodulin-dependent protein kinase Ia kinase. Phosphorylation of threonine 196 is essential for activation.

Purified pig brain Ca(2+)-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A.