D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333–372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIα and RIIα. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Gα protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

MLCK-A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway

Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca2+/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A’s activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when Dictyostelium cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.

Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C

The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of ≈20-kDa C-terminal fragments (CTF) and ≈30-kDa N-terminal fragments [Thinakaran, G., et al. (1996) Neuron 17, 181–190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the ≈20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G., et al. (1996) Neuron 17, 181–190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an in vivo substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.

TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines

We have previously identified a cellular protein kinase activity termed TAK that specifically associates with the HIV types 1 and 2 Tat proteins. TAK hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II in vitro in a manner believed to activate transcription [Herrmann, C. H. & Rice, A. P. (1995) J. Virol. 69, 1612–1620]. We show here that the catalytic subunit of TAK is a known human kinase previously named PITALRE, which is a member of the cyclin-dependent family of proteins. We also show that TAK activity is elevated upon activation of peripheral blood mononuclear cells and peripheral blood lymphocytes and upon differentiation of U1 and U937 promonocytic cell lines to macrophages. Therefore, in HIV-infected individuals TAK may be induced in T cells following activation and in macrophages following differentiation, thus contributing to high levels of viral transcription and the escape from latency of transcriptionally silent proviruses.

Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIα regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.

Increased IGF-II protein affects p57kip2 expression in vivo and in vitro: Implications for Beckwith–Wiedemann syndrome

In both human and mouse, the Igf2 gene, localized on chromosomes 11 and 7, respectively, is expressed from the paternally inherited chromosome in the majority of tissues. Insulin-like growth factor-II (IGF-II) plays an important role in embryonic growth, and aberrant IGF2 expression has been documented in several human pathologies, such as Beckwith–Wiedemann syndrome (BWS), and a wide variety of tumors. Human and mouse genetic data strongly implicate another gene, CDKN1C (p57kip2), located in the same imprinted gene cluster on human chromosome II, in BWS. p57KIP2 is a cyclin-dependent kinase inhibitor and is required for normal mouse embryonic development. Mutations in CDKN1C (p57kip2) have been identified in a small proportion of patients with BWS, and removal of the gene from mice by targeted mutagenesis produces a phenotype with elements in common with this overgrowth syndrome. Patients with BWS with biallelic expression of IGF2 or with a CDKN1C (p57kip2) mutation, as well as overlapping phenotypes observed in two types of mutant mice, the p57kip2 knockout and IGF-II-overexpressing mice, strongly suggest that the genes may act in a common pathway of growth control in situations where Igf2 expression is abnormal. Herein, we show that p57kip2 expression is reduced on IGF-II treatment of primary embryo fibroblasts in a dose-dependent manner. In addition, p57kip2 expression is down-regulated in mice with high serum levels of IGF-II. These data suggest that the effects of increased IGF-II in BWS may, in part, be mediated through a decrease in p57kip2 gene expression.

Neuropeptide Y (NPY) potentiates phenylephrine-induced mitogen-activated protein kinase activation in primary cardiomyocytes via NPY Y5 receptors

Neuropeptide Y (NPY) has been shown to participate in the cardiovascular response mediated by the sympathetic system. In this report, we investigate the growth factor properties of NPY on cardiac myocytes. Mitogen-activated protein kinases (MAPK) are key signaling molecules in the transduction of trophic signals. Therefore, the role of NPY in inducing MAPK activation was studied in mouse neonatal cardiomyocytes. Exposure of neonatal cardiomyocytes to either NPY, phenylephrine, or angiotensin II induces a rapid phosphorylation of the extracellular responsive kinase, the c-jun N-terminal kinase, and the p38 kinase as well as an activation of protein kinase C (PKC). Moreover, NPY potentiates phenylephrine-induced MAPK and PKC stimulation. In contrast, NPY has no synergistic effect on angiotensin II-stimulated MAPK phosphorylation or PKC activity. NPY effects are pertussis toxin-sensitive and calcium-independent and are mediated by NPY Y5 receptors. Taken together, these results suggest that NPY, via Gi protein-coupled NPY Y5 receptors, could participate in the development of cardiac hypertrophy during chronic sympathetic stimulation by potentiating α-adrenergic signals.

Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain

A sensitive assay using biotinylated ubiquitin revealed extensive ubiquitination of the large subunit of RNA polymerase II during incubations of transcription reactions in vitro. Phosphorylation of the repetitive carboxyl-terminal domain of the large subunit was a signal for ubiquitination. Specific inhibitors of cyclin-dependent kinase (cdk)-type kinases suppress the ubiquitination reaction. These kinases are components of transcription factors and have been shown to phosphorylate the carboxyl-terminal domain. In both regulation of transcription and DNA repair, phosphorylation of the repetitive carboxyl-terminal domain by kinases might signal degradation of the polymerase.

Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane

Accumulating evidence suggests that the mitochondrial molecular chaperone heat shock protein 60 (hsp60) also can localize in extramitochondrial sites. However, direct evidence that hsp60 functions as a chaperone outside of mitochondria is presently lacking. A 60-kDa protein that is present in the plasma membrane of a human leukemic CD4+ CEM-SS T cell line and is phosphorylated by protein kinase A (PKA) was identified as hsp60. An 18-kDa plasma membrane-associated protein coimmunoprecipitated with hsp60 and was identified as histone 2B (H2B). Hsp60 physically associated with H2B when both molecules were in their dephospho forms. By contrast, PKA-catalyzed phosphorylation of both hsp60 and H2B caused dissociation of H2B from hsp60 and loss of H2B from the plasma membrane of intact T cells. These results suggest that (i) hsp60 and H2B can localize in the T cell plasma membrane; (ii) hsp60 functions as a molecular chaperone for H2B; and (iii) PKA-catalyzed phosphorylation of both hsp60 and H2B appears to regulate the attachment of H2B to hsp60. We propose a model in which phosphorylation/dephosphorylation regulates chaperoning of H2B by hsp60 in the plasma membrane.

Interaction of a transcriptional repressor with the RNA polymerase II holoenzyme plays a crucial role in repression

The yeast transcriptional repressor Tup1, tethered to DNA, represses to strikingly different degrees transcription elicited by members of two classes of activators. Repression in both cases is virtually eliminated by mutation of either member of the cyclin-kinase pair Srb10/11. In contrast, telomeric chromatin affects both classes of activators equally, and in neither case is that repression affected by mutation of Srb10/11. In vitro, Tup1 interacts with RNA polymerase II holoenzyme bearing Srb10 as well as with the separated Srb10. These and other findings indicate that at least one aspect of Tup1's action involves interaction with the RNA polymerase II holoenzyme.

Novel Nuclear Signaling Pathway Mediates Activation of Fibroblast Growth Factor-2 Gene by Type 1 and Type 2 Angiotensin II Receptors

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1′,3′-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

Antagonistic forces generated by myosin II and cytoplasmic dynein regulate microtubule turnover, movement, and organization in interphase cells

Photoactivation of caged fluorescent tubulin was used mark the microtubule (MT) lattice and monitor MT behavior in interphase cells. A broadening of the photoactivated region occurred as MTs moved bidirectionally. MT movement was not inhibited when MT assembly was suppressed with nocodazole or Taxol; MT movement was suppressed by inhibition of myosin light chain kinase with ML7 or by a peptide inhibitor. Conversely, MT movement was increased after inhibition of cytoplasmic dynein with the antibody 70.1. In addition, the half-time for MT turnover was decreased in cells treated with ML7. These results demonstrate that myosin II and cytoplasmic dynein contribute to a balance of forces that regulates MT organization, movement, and turnover in interphase cells.

An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells.

Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.

A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus.

The cDNA corresponding to a fourth species of diacylglycerol (DG) kinase (EC 2.7.1.107) was isolated from cDNA libraries of rat retina and brain. This cDNA encoded a 929-aa, 104-kDa polypeptide termed DGK-IV. DGK-IV was different from previously identified mammalian DG kinase species, DGK-I, DGK-II, and DGK-III, in that it contained no EF-hand motifs but did contain four ankyrin-like repeats at the carboxyl terminus. These structural features of DGK-IV closely resemble the recently cloned, eye-specific DG kinase of Drosophila that is encoded by the retinal degeneration A (rdgA) gene. However, DGK-IV was expressed primarily in the thymus and brain with relatively low expression in the eye and intestine. Furthermore, the primary structure of the DGK-IV included a nuclear targeting motif, and immunocytochemical analysis revealed DGK-IV to localize in the nucleus of COS-7 cells transfected with the epitope-tagged cDNA, suggesting an involvement of DGK-IV in intranuclear processes.

Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase.

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.