A protein kinase encoded by the t complex responder gene causes non-mendelian inheritance

Males heterozygous for the t-haplotype form of mouse chromosome 17 preferentially transmit the t-chromosome to their progeny. Several distorter/sterility loci carried on the t-haplotype together impair flagellar function in all spermatozoa whereas the responder, Tcr, rescues t-sperm but not wild-type sperm. Thus, t-sperm have an advantage over wild-type sperm in fertilizing egg cells. We have isolated Tcr by positional cloning and show that it is a member of a novel protein kinase gene family, designated Smok, which is expressed late during spermiogenesis. Smok kinases are components of a signal cascade which may control sperm motility. Tcr has a reduced kinase activity, which may allow it to counterbalance a signalling impairment caused by the distorter/sterility loci. Tcr transgene constructs cause non-mendelian transmission of chromosomes on which they are carried, which leads to sex-ratio distortion when Tcr cosegregates with the Y chromosome.     

A mutation altering the function of a carbohydrate binding protein blocks cell-cell cohesion in developing Dictyostelium discoideum.

In Dictyostelium discoideum, carbohydrate binding proteins (CBPs) or lectins have been implicated in the molecular basis of cellular cohesion. To determine the role of these CBPs, we have attempted to isolate structural gene mutants in which the CBPs have a defective affinity for carbohydrate ligands. We now report the isolation of a spontaneous, cross-reacting material (CRM) mutant which is non-cohesive and fails to develop. The mutant seems to have a defect in the structural gene for one of the two developmentally regulated carbohydrate binding proteins (CBP-26), which renders it unable to bind to galactose-containing ligands. The fact that wild-type cells interact with the mutant and carry it through development strongly supports a model of cell-cell interaction in which cohesion is mediated by complementary molecules.     

A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) is a major pathogen in immunosuppressed individuals, including patients with acquired immune deficiency syndrome. The nucleoside analogue ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)-guanine) is one of the few drugs available to treat HCMV infections, but resistant virus is a growing problem in the clinic and there is a critical need for new drugs. The study of ganciclovir-resistant mutants has indicated that the selective action of ganciclovir depends largely on virus-controlled phosphorylation in HCMV-infected cells. The enzyme(s) responsible have not been identified. Here we report that the HCMV gene UL97, whose predicted product shares regions of homology with protein kinases, guanylyl cyclase and bacterial phosphotransferases, controls phosphorylation of ganciclovir in HCMV-infected cells. A four-amino-acid deletion of UL97 in a conserved region, which in cyclic AMP-dependent protein kinase participates in substrate recognition, causes impaired ganciclovir phosphorylation. The implications of these results for antiviral drug development and drug resistance are discussed.     

Application of phorbol ester to mouse skin causes a rapid and sustained loss of protein kinase C

It is now widely accepted that tumour-promoting phorbol esters activate a Ca2+- and phospholipid-dependent protein kinase (protein kinase C) both in vitro and in intact cells, and that the kinase represents a major cellular phorbol ester-binding protein. The phorbol esters act as analogues of diacylglycerol, a natural regulator of protein kinase C, and stabilize the membrane-association of the kinase. Although other molecular targets may exist, protein kinase C activation is probably important in mediating the diverse responses of cultured cells to phorbol esters and in promoting in vivo tumours. The enzyme comprises a family of closely related proteins and has been detected in extracts from mouse epidermal cells, the likely targets for two-stage carcinogenesis in mouse skin. In this report we show that application of a single dose of TPA (12-O-tetradecanoyl phorbol-13-acetate) to mouse skin results in a rapid and complete loss of protein kinase C activity which is maintained for 3-4 days. This is associated with a loss of immunologically detectable protein kinase C and the accumulation of a smaller protein detectable by antibodies recognizing the regulatory domain of protein kinase C.     

盘基网柄菌发育期间蛞蝓体的形态学研究

用吖啶橙和Hoechst 33342两种活体荧光染料,通过荧光显微镜观察,来了解盘基网柄菌多细胞发育期间蛞蝓体阶段中出现的细胞分化及凋亡特点.结果发现:随着发育进程,将发育成柄细胞的前柄细胞先被染成蓝绿色,然后被染成绿色,再成橙色,最后为无色,这些分别表示了前柄细胞不同的凋亡阶段.在蛞蝓体后期其内部出现一条"通道".得出结论,蛞蝓体是盘基网柄菌细胞分化和衰退的起始阶段,其形态发生了巨大的变化.前柄细胞从蛞蝓体前期开始凋亡,但并不是马上死亡,而是一个渐进的凋亡过程.     

A receptor kinase gene regulating symbiotic nodule development

Leguminous plants are able to establish a nitrogen-fixing symbiosis with soil bacteria generally known as rhizobia. Metabolites exuded by the plant root activate the production of a rhizobial signal molecule, the Nod factor, which is essential for symbiotic nodule development. This lipo-chitooligosaccharide signal is active at femtomolar concentrations, and its structure is correlated with host specificity of symbiosis, suggesting the involvement of a cognate perception system in the plant host. Here we describe the cloning of a gene from Medicago sativa that is essential for Nod-factor perception in alfalfa, and by genetic analogy, in the related legumes Medicago truncatula and Pisum sativum. The identified 'nodulation receptor kinase', NORK, is predicted to function in the Nod-factor perception/transduction system (the NORK system) that initiates a signal cascade leading to nodulation. The family of 'NORK extracellular-sequence-like' (NSL) genes is broadly distributed in the plant kingdom, although their biological function has not been previously ascribed. We suggest that during the evolution of symbiosis an ancestral NSL system was co-opted for transduction of an external ligand, the rhizobial Nod factor, leading to development of the symbiotic root nodule.     

Phosducin is a protein kinase A-regulated G-protein regulator.

Signal transduction by G-protein-coupled receptors is regulated by various mechanisms acting at the receptor level; those studied most thoroughly are from the beta-adrenergic receptor/Gs/adenylyl cyclase system. We report here a regulatory mechanism occurring at the level of the G proteins themselves. A protein with M(r) 33,000 that inhibits Gs-GTPase activity was purified from bovine brain. This protein is very similar or identical to phosducin, a protein previously thought to be specific for retina and pineal gland. Recombinant phosducin inhibited the GTPase activity of several G proteins, and also inhibited Gs-mediated adenylyl cyclase activation. Blockade of its inhibitory effects by protein kinase A suggests that phosducin may be part of a complex regulatory network controlling G-protein-mediated signalling.     

A mutant protein kinase C that can transform fibroblasts

Expression of normal protein kinase C (PKC) isoenzymes in fibroblasts has been shown to alter growth regulation but has failed to induce complete transformation of the recipient cells. Here we report on a murine ultraviolet-induced fibrosarcoma cell line which has an unusual PKC subcellular distribution with 87% of the PKC activity associated with the membrane. We have cloned and sequenced the alpha-PKC complementary DNA from ultraviolet-induced-fibrosarcoma cells and from mouse Balb/c brain and found four point mutations in the fibrosarcoma PKC, of which three are in the highly conserved regulatory domain and one is in the conserved region of the catalytic domain. Expression of this mutant alpha-PKC gene in normal Balb/c 3T3 fibroblasts results in a fibrosarcoma-like PKC membrane localization and in cell transformation, as judged by their formation of dense foci, anchorage-independent growth and ability to induce solid tumours when inoculated into nude mice. By contast, transfectants expressing the normal alpha-PKC cDNA do not display a morphology typical of malignant transformed cells and fail to induce tumours in vivo. These findings demonstrate that point mutations in the primary structure of PKC modulate enzyme function and are responsible for inducing oncogenicity.     

Hedgehog signalling activity of Smoothened requires phosphorylation by protein kinase A and casein kinase I

The Hedgehog (Hh) family of secreted proteins governs cell growth and patterning in animal development. The Hh signal is transduced by the seven-transmembrane protein Smoothened (Smo); however, the mechanism by which Smo is regulated remains largely unknown. Here we show that protein kinase A (PKA) and casein kinase I (CKI) regulate Smo cell-surface accumulation and activity in response to Hh. Blocking PKA or CKI activity in the Drosophila wing disc prevents Hh-induced Smo accumulation and attenuates pathway activity, whereas increasing PKA activity promotes Smo accumulation and pathway activation. We show that PKA and CKI phosphorylate Smo at several sites, and that phosphorylation-deficient forms of Smo fail to accumulate on the cell surface and are unable to transduce the Hh signal. Conversely, phosphorylation-mimicking Smo variants show constitutive cell-surface expression and signalling activity. Furthermore, we find that the levels of Smo cell-surface expression and activity correlate with its levels of phosphorylation. Our data indicate that Hh induces progressive Smo phosphorylation by PKA and CKI, leading to elevation of Smo cell-surface levels and signalling activity.     

A chemical switch for inhibitor-sensitive alleles of any protein kinase

Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.     

The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways

Cyclic adenosine 3', 5'-monophosphate (cAMP) is a ubiquitous mediator of intracellular signalling events. It acts principally through stimulation of cAMP-dependent protein kinases (PKAs) but also activates certain ion channels and guanine nucleotide exchange factors (Epacs). Metabolism of cAMP is catalysed by phosphodiesterases (PDEs). Here we identify a cAMP-responsive signalling complex maintained by the muscle-specific A-kinase anchoring protein (mAKAP) that includes PKA, PDE4D3 and Epac1. These intermolecular interactions facilitate the dissemination of distinct cAMP signals through each effector protein. Anchored PKA stimulates PDE4D3 to reduce local cAMP concentrations, whereas an mAKAP-associated ERK5 kinase module suppresses PDE4D3. PDE4D3 also functions as an adaptor protein that recruits Epac1, an exchange factor for the small GTPase Rap1, to enable cAMP-dependent attenuation of ERK5. Pharmacological and molecular manipulations of the mAKAP complex show that anchored ERK5 can induce cardiomyocyte hypertrophy. Thus, two coupled cAMP-dependent feedback loops are coordinated within the context of the mAKAP complex, suggesting that local control of cAMP signalling by AKAP proteins is more intricate than previously appreciated.     

Inhibitors of protein kinase C

Protein kinase catalyzes the transfer of the γ-phosphoryl group from ATP to the hydroxyl groups o fprotein side chains, which plays critical roles in signal transduction pathways by transmitting extracellular signals across the plasma membrane and nuclear membrane to the destination sites in the cytoplasm and the nucleus. Protein kinase C (PKC) is a superfamily of phospholipid-dependent Ser/Thr kinase. There are at least 12 isozymes in PKC family.They are distributed in different tissues and play different roles in physiological processes. On account of their concern with a variety of pathophysiologic states, such as cancer,inflammatory conditions, autoimmune disorder, and cardiac diseases, the inhibitors, which can inhibit the activity of PKC and the interaction of cytokine with receptor, and interfere signal transduction pathway, may be candidates of therapeutic drugs. Therefore, intense efforts have been made to develop specific protein kinase inhibitors as biological tools and therapeutic agents. This article reviews the recent development of some of PKC inhibitors based on their interaction with different conserved domains and different inhibition mechanisms.     

Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.     

Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes

Hormones mobilize intracellular second messengers and initiate signalling cascades involving protein kinases and phosphatases, which are often spatially compartmentalized by anchoring proteins to increase signalling specificity. These scaffold proteins may themselves be modulated by hormones. In adipocytes, stimulation of beta-adrenergic receptors increases cyclic AMP levels and activates protein kinase A (PKA), which stimulates lipolysis by phosphorylating hormone-sensitive lipase and perilipin. Acute insulin treatment activates phosphodiesterase 3B, reduces cAMP levels and quenches beta-adrenergic receptor signalling. In contrast, chronic hyperinsulinaemic conditions (typical of type 2 diabetes) enhance beta-adrenergic receptor-mediated cAMP production. This amplification of cAMP signalling is paradoxical because it should enhance lipolysis, the opposite of the known short-term effect of hyperinsulinaemia. Here we show that in adipocytes, chronically high insulin levels inhibit beta-adrenergic receptors (but not other cAMP-elevating stimuli) from activating PKA. We measured this using an improved fluorescent reporter and by phosphorylation of endogenous cAMP-response-element binding protein (CREB). Disruption of PKA scaffolding mimics the interference of insulin with beta-adrenergic receptor signalling. Chronically high insulin levels may disrupt the close apposition of beta-adrenergic receptors and PKA, identifying a new mechanism for crosstalk between heterologous signal transduction pathways.     

Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase.

Leptin is a hormone secreted by adipocytes that plays a pivotal role in regulating food intake, energy expenditure and neuroendocrine function. Leptin stimulates the oxidation of fatty acids and the uptake of glucose, and prevents the accumulation of lipids in nonadipose tissues, which can lead to functional impairments known as "lipotoxicity". The signalling pathways that mediate the metabolic effects of leptin remain undefined. The 5'-AMP-activated protein kinase (AMPK) potently stimulates fatty-acid oxidation in muscle by inhibiting the activity of acetyl coenzyme A carboxylase (ACC). AMPK is a heterotrimeric enzyme that is conserved from yeast to humans and functions as a 'fuel gauge' to monitor the status of cellular energy. Here we show that leptin selectively stimulates phosphorylation and activation of the alpha2 catalytic subunit of AMPK (alpha2 AMPK) in skeletal muscle, thus establishing a previously unknown signalling pathway for leptin. Early activation of AMPK occurs by leptin acting directly on muscle, whereas later activation depends on leptin functioning through the hypothalamic-sympathetic nervous system axis. In parallel with its activation of AMPK, leptin suppresses the activity of ACC, thereby stimulating the oxidation of fatty acids in muscle. Blocking AMPK activation inhibits the phosphorylation of ACC stimulated by leptin. Our data identify AMPK as a principal mediator of the effects of leptin on fatty-acid metabolism in muscle.