Bone morphogenetic protein function is required for terminal differentiation of the heart but not for early expression of cardiac marker genes.

To examine potential roles for bone morphogenetic proteins (BMPs) in cardiogenesis, we used intracellular BMP inhibitors to disrupt this signaling cascade in Xenopus embryos. BMP-deficient embryos showed endodermal defects, a reduction in cardiac muscle-specific gene expression, a decrease in the number of cardiomyocytes and cardia bifida. Early expression of markers of endodermal and precardiac fate, however, was not perturbed. Heart defects were observed even when BMP signal transduction was blocked only in cells that contribute primarily to endodermal, and not cardiac fates, suggesting a non-cell autonomous function. Our results suggest that BMPs are not required for expression of early transcriptional regulators of cardiac fate but are essential for migration and/or fusion of the heart primordia and cardiomyocyte differentiation.

CaM kinase IV regulates lineage commitment and survival of erythroid progenitors in a non-cell-autonomous manner.

Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

Agonist-dependent desensitization of the kappa opioid receptor by G protein receptor kinase and beta-arrestin.

We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.

Cloning, chromosomal mapping, and regulatory properties of the human type 9 adenylyl cyclase (ADCY9).

The type 9 adenylyl cyclase (AC9) is a widely distributed adenylyl cyclase that was originally cloned from a mouse cDNA library. Here we report the cloning, chromosomal mapping, and regulatory properties of human AC9 (HGMW-approved symbol ADCY9). Although the human AC9 sequence shows 86% homology with mouse AC9, divergence at the C2a/C2b junction results in an alternative C2b amino acid sequence. In situ hybridization localized the human AC9 gene to both human and mouse chromosomes 16. AC9 mRNA is present in all tissues examined, with the highest levels found in skeletal muscle, heart, and brain. To characterize the regulatory properties of human AC9 in vivo, the enzyme was expressed in HEK-293 cells. Human AC9 is stimulated by beta-adrenergic receptor activation but is insensitive to forskolin, Ca2+ and somatostatin. In contrast to mouse AC9, the activity of human AC9 is unaffected by inhibitors of calcineurin. These data emphasize the importance of determining the regulatory properties of human adenylyl cyclases.

Calcium/calmodulin-dependent protein kinase kinase: identification of regulatory domains.

We recently cloned a calmodulin-dependent protein kinase kinase (CaM-KK) which phosphorylates and activates CaM-KI and CaM-KIV [Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324]. In the present study, we have identified its regulatory CaM-binding and autoinhibitory domains (CBD and AID, respectively) using a series of COOH-terminal truncations and site-directed mutants expressed in COS-7 cells. Truncation mutant CaM-KK1-463 activated CaM-KIV and bound CaM similar to wild-type enzyme (CaM-KK1-505); CaM-KK1-448 did not bind CaM and was largely inactive; and CaM-KK1-434 also did not bind CaM but activated a CaM-independent mutant of CaM-KIV in the absence of Ca2+/CaM. Substitution of triple negative charges (Asp) at positions 455RKR, 448ILV, or 443SWT blocked CaM binding and suppressed by 70-90% CaM-KK activities. Mutants 438VKL and 435KNS to DDD exhibited partial Ca2+/CaM-independent activities. These results identify overlapping AID and CBD between residues 430 and 460 in CaM-KK, similar to other CaM-Ks. Consistent with this assignment, the synthetic peptide corresponding to residues 438-463 bound CaM in a Ca2+-dependent manner with a Kd in the low nanomolar range. Furthermore, phosphorylation by cAMP-kinase of Ser458 at the COOH-terminus of the CBD in CaM-KK, which suppresses subsequent CaM binding [Wayman, G., Tokumitsu, H., and Soderling, T. R. (1997) J. Biol. Chem. 272, 16073-16076], was blocked by prior binding of Ca2+/CaM to CaM-KK.

Inhibitory cross-talk by cAMP kinase on the calmodulin-dependent protein kinase cascade.

The calmodulin-dependent kinase (CaM-K) cascade, a Ca2+-triggered system involving phosphorylation and activation of CaM-KI and CaM-KIV by CaM kinase kinase (CaM-KK), regulates transcription through direct phosphorylation of transcription factors such as cAMP response element-binding protein. We have shown previously that activated CaM-KIV can activate the mitogen-activated protein kinases (Enslen, H., Tokumitsu, H., Stork, P. J. S., Davis, R. J., and Soderling, T. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10803-10808), and the present paper describes a novel regulatory cross-talk between cAMP kinase (PKA) and CaM-KK. PKA gave rapid phosphorylation in vitro and in cells of recombinant CaM-KK, resulting in 50-75% inhibition of CaM-KK activity, part of which was due to suppression of CaM-binding by phosphorylation of Ser458 in the CaM-binding domain. However, the Ser458 --> Ala mutant, or a truncation mutant in which the CaM-binding and autoinhibitory domains were deleted, was still partially suppressed by PKA-mediated phosphorylation. The second inhibitory site was identified as Thr108 by site-specific mutagenesis. Treatments of COS-7, PC12, hippocampal, or Jurkat cells with the PKA activators forskolin or isoproterenol gave 30-90% inhibition of either endogenous or transfected CaM-KK and/or CaM-KIV activities. These results demonstrate that the CaM kinase cascade is negatively regulated in cells by the cAMP/PKA pathway.

The Ca2+/calmodulin-dependent kinase type IV is involved in the CD5-mediated signaling pathway in human T lymphocytes.

The CD5 receptor on T lymphocytes is involved in T cell activation and T-B cell interactions. In the present study, we have characterized the signaling pathways induced by anti-CD5 stimulation in human T lymphocytes. In T lymphocytes, anti-CD5 co-stimulation enhances the phytohemagglutinin/anti-CD28-induced interleukin-2 (IL-2) mRNA accumulation 1.6-fold and IL-2 protein secretion 2. 2-fold, whereby the up-regulation is mediated at both the transcriptional and post-transcriptional level. The CD5 signaling pathway up-regulates the IL-2 gene expression by increasing the DNA binding and transactivation activity of activator protein 1 but affects none of the other transcription factors like nuclear factor of activated T cells, nuclear factor kappaB, Oct, and CD28-responsive complex/nuclear factor of mitogen-activated T cells involved in the regulation of the IL-2 promoter activity. The CD5-induced increase of the activator protein 1 activity is mediated through the activation of calcium/calmodulin-dependent (CaM) kinase type IV, and is independent of the activation of mitogen-activated protein kinases Jun N-terminal kinase, extracellular signal-regulated kinase, and p38/Mpk2, and calcium/calmodul-independent kinase type II. The expression of a dominant negative mutant of CaM kinase IV in T lymphocytes transfected with an IL-2 promoter-driven reporter construct completely abrogates the response to CD5 stimulation, indicating that CaM kinase IV is essential to the CD5 signaling pathway. In addition, it is demonstrated that calcium/calmodulin-dependent kinase type IV is also involved in the stabilization of the IL-2 transcripts, which is observed after co-stimulation of phytohemagglutinin/anti-CD28 activated T lymphocytes with anti-CD5.

Regulation of type I adenylyl cyclase by calmodulin kinase IV in vivo.

Type I adenylyl cyclase is a neurospecific enzyme that is stimulated by Ca2+ and calmodulin (CaM). This enzyme couples the Ca2+ and cyclic AMP (cAMP) regulatory systems in neurons, and it may play an important role for some forms of synaptic plasticity. Mutant mice lacking type I adenylyl cyclase show deficiencies in spatial memory and altered long-term potentiation (Z. Wu, S. A. Thomas, Z. Xia, E. C. Villacres, R. D. Palmiter, and D. R. Storm, Proc. Natl. Acad. Sci. USA 92:220-224, 1995). Although type I adenylyl cyclase is synergistically stimulated by Ca2+ and G-protein-coupled receptors in vivo, very little is known about mechanisms for inhibition of the enzyme. Here, we report that type I adenylyl cyclase is inhibited by CaM kinase IV in vivo. Expression of constitutively active or wild-type CaM kinase IV inhibited Ca2+ stimulation of adenylyl cyclase activity without affecting basal or forskolin-stimulated activity. Type I adenylyl cyclase has two CaM kinase IV consensus phosphorylation sequences near its CaM binding domain at Ser-545 and Ser-552. Conversion of either serine to alanine by mutagenesis abolished CaM kinase IV inhibition of adenylyl cyclase. This suggests that the activity of this enzyme may be directly inhibited by CaM kinase IV phosphorylation. Type VIII adenylyl cyclase, another enzyme stimulated by CaM, was not inhibited by CaM kinase II or IV. We propose that CaM kinase IV may function as a negative feedback regulator of type I adenylyl cyclase and that CaM kinases may regulate cAMP levels in some cells.

Hormone stimulation of type III adenylyl cyclase induces Ca2+ oscillations in HEK-293 cells.

Various forms of cross-talk between the Ca2+ and cAMP signal transduction systems can occur in animal cells depending upon the types of adenylyl cyclases present. Here, we report that Ca2+ oscillations can be generated by hormone stimulation of type III adenylyl cyclase expressed in HEK-293 cells. These Ca2+ oscillations are apparently due to the unique regulatory features of type III adenylyl cyclase, which is stimulated by hormones and inhibited by elevated Ca2+ in vivo. Ca2+ oscillations were generated by glucagon, isoproterenol, or forskolin stimulation of type III adenylyl cyclase and were dependent upon the activity of cAMP- and calmodulin-dependent protein kinases. Ca2+ oscillations were not solely dependent upon cAMP increases since dibutyryl cAMP or (Sp)-cAMP did not stimulate Ca2+ oscillations. We hypothesize that stimulation of type III adenylyl cyclase leads to increased cAMP, activation of inositol 1,4,5-trisphosphate receptors, and elevation of intracellular Ca2+. As free Ca2+ increases, type III adenylyl cyclase activity is attenuated by CaM kinase(s) and intracellular cAMP levels decrease. When cAMP levels drop below a threshold level, the inositol 1,4,5-trisphosphate receptor is dephosphorylated and Ca2+ is resequestered. This cycle is repeated if type III adenylyl cyclase is chronically exposed to an activator. This unique mechanism for generation of Ca2+ oscillations in cells is distinct from others documented in the literature.

Ca2+ inhibition of type III adenylyl cyclase in vivo.

Type III adenylyl cyclase is stimulated by beta-adrenergic agonists and glucagon in vitro and in vivo, but not by Ca2+ and calmodulin. However, the enzyme is stimulated by Ca2+ and calmodulin in vitro when it is concomitantly activated by the guanyl nucleotide stimulatory protein Gs (Choi, E. J., Xia, Z., and Storm, D. R. (1992a) Biochemistry 31, 6492-6498). Here, we examined regulation of type III adenylyl cyclase by Gs-coupled receptors and intracellular Ca2+ in vivo. Surprisingly, intracellular Ca2+ inhibited hormone-stimulated type III adenylyl cyclase activity. Submicromolar concentrations of intracellular free Ca2+, which stimulated type I adenylyl cyclase, inhibited glucagon- or isoproterenol-stimulated type III adenylyl cyclase. Inhibition of type III adenylyl cyclase by intracellular Ca2+ was not mediated by Gi, cAMP-dependent protein kinase, or protein kinase C. However, an inhibitor of CaM kinases antagonized Ca2+ inhibition of the enzyme, and coexpression of constitutively activated CaM kinase II completely inhibited isoproterenol-stimulated type III adenylyl cyclase activity. We propose that Ca2+ inhibition of type III adenylyl cyclase may serve as a regulatory mechanism to attenuate hormone-stimulated cAMP levels in some tissues.

Synergistic activation of the type I adenylyl cyclase by Ca2+ and Gs-coupled receptors in vivo.

The type I adenylyl cyclase is directly stimulated by Ca2+ and calmodulin in vivo (Choi, E. J., Wong, S. T., Hinds, T. R. and Storm, D. R. (1992) J. Biol. Chem. 267, 12440-12442; Wu, Z., Wong, S. T., and Storm, D. R. (1993) J. Biol. Chem. 268, 23766-23768). In this study, we examined the sensitivity of the type I adenylyl cyclase expressed in HEK-293 cells to beta-adrenergic agonists or glucagon when intracellular Ca2+ was elevated by Ca2+ ionophore or carbachol. Although previous studies have shown that this enzyme can be directly stimulated by activated Gs in vitro, we demonstrate that it is not stimulated by Gs-coupled receptors in vivo. However, the enzyme was stimulated by Gs-coupled receptors in vivo when it was activated by intracellular Ca2+. For example, the Ca2+ ionophore A23187 stimulated the enzyme 3 +/- 0.5-fold (n = 9) and isoproterenol alone did not stimulate the enzyme, but the combination of the two stimulated type I adenylyl cyclase 13 +/- 2-fold (n = 9) in vivo. Similarly, 500 nM glucagon alone did not stimulate the enzyme but the combination of A23187 and glucagon activated the enzyme 90 +/- 8-fold (n = 4). Synergistic stimulation of type I adenylyl cyclase activity was also obtained with combinations of carbachol and isoproterenol or glucagon. This phenomenon was not observed with a mutant enzyme that is insensitive to Ca2+ and calmodulin, suggesting that conformational changes caused by binding of calmodulin to the type I adenylyl cyclase enhance binding or coupling to activated Gs. These data illustrate that this adenylyl cyclase can couple Ca2+ and neurotransmitter signals to generate optimal cAMP levels, a property of the enzyme that may be important for its role in learning and memory in mammals.