The Tumor Suppressor, PTEN/MMAC1, Dephosphorylates the Lipid Second Messenger, Phosphatidylinositol 3,4,5-Trisphosphate

Phagocytosis of pathogens by macrophages initiates the innate immune response, which in turn orchestrates the adaptive response. In order to discriminate between infectious agents and self, macrophages have evolved a restricted number of phagocytic receptors, like the mannose receptor, that recognize conserved motifs on pathogens. Pathogens are also phagocytosed by complement receptors after relatively nonspecific opsonization with complement and by Fc receptors after specific opsonization with antibodies. All these receptors induce rearrangements in the actin cytoskeleton that lead to the internalization of the particle. However, important differences in the molecular mechanisms underlying phagocytosis by different receptors are now being appreciated. These include differences in the cytoskeletal elements that mediate ingestion, differences in vacuole maturation, and differences in inflammatory responses. Infectious agents, such as M. tuberculosis, Legionella pneumophila, and Salmonella typhimurium, enter macrophages via heterogeneous pathways and modify vacuolar maturation in a manner that favors their survival. Macrophages also play an important role in the recognition and clearance of apoptotic cells; a notable feature of this process is the absence of an inflammatory response.

Mechanisms of phagocytosis in macrophages

Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

Small GTP-Binding Proteins

There are ample genetic and laboratory studies that suggest the PI3K-Akt pathway is vital to the growth and survival of cancer cells. Inhibitors targeting this pathway are entering the clinic at a rapid pace. In this Review, the therapeutic potential of drugs targeting PI3K-Akt signalling for the treatment of cancer is discussed. I focus on the advantages and drawbacks of different treatment strategies for targeting this pathway, the cancers that might respond best to these therapies and the challenges and limitations that confront their clinical development.

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Targeting PI3K signalling in cancer: opportunities, challenges and limitations.

A family of cyclin-dependent kinases (CDKs) cooperatively regulates mammalian cell cycle progression (for review, see Sherr 1993). During G1 phase, D-type cyclins (D1, D2, and D3) are synthesized and assemble with either CDK4 or CDK6 in response to growth factor stimulation, thereby generating active holoenzymes that help inactivate the growth-suppressive function of the retinoblastoma protein (Rb) through its phosphorylation (for review, see Weinberg 1995). Cyclin D holoenzyme complexes also titrate CDK inhibitors, such as p27Kip1 and p21Cip1, facilitating the activation of cyclin E-CDK2 and subsequent entry into the DNA synthetic phase of the cell cycle (for review, see Sherr and Roberts 1995).

Ras-mediated pathways are important for cyclin D1 induction and its assembly with CDKs. Overexpression of activated oncogenic Ras alleles, but not wild-type Ras, initiates DNA synthesis independently of growth factor stimulation (Feramisco et al. 1984). Conversely, microinjection of antibodies that inactivate Ras or introduction of certain dominant-negative Ras alleles can block S-phase entry induced by mitogens (Mulcahy et al. 1985; Mittnacht et al. 1997; Peeper et al. 1997). Both cyclin D1 expression and assembly require the sequential activities of Raf1, mitogen-activated protein kinase-kinases (MEK1 and MEK2), and the sustained activation of extracellular signal-regulated protein kinases (ERKs; Albanese et al. 1995; Lavoie et al. 1996; Winston et al. 1996; Aktas et al. 1997; Kerkhoff and Rapp 1997; Weber et al. 1997; Cheng et al. 1998).

In turn, cyclin D1 degradation is mediated by phosphorylation-triggered, ubiquitin-dependent proteolysis (Diehl et al. 1997). Polyubiquitination of protein substrates involves the sequential action of three distinct enzymes termed E1, E2 (UBC; ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase; Ciechanover 1994; King et al. 1996). Specificity of substrate recognition is dependent on several factors including E2 and E3 selectivity (King et al. 1996; Skowyra et al. 1997; Renny-Feldman et al. 1997), recognition motifs within the target proteins themselves (Glotzer et al. 1991), and, in some cases, a requirement for phosphorylation of specific residues within the substrate (Deshaies et al. 1995; Clurman et al. 1996; Lanker et al. 1996; Won et al. 1996). Ubiquitin-dependent degradation of cyclin D1 requires phosphorylation of a specific threonine residue (Thr-286) located near the protein carboxyl terminus, and this phosphorylation is not mediated by cyclin D-dependent kinases themselves (Diehl et al. 1997). Because the kinase that phosphorylates this residue has not yet been identified, it remains unclear whether cyclin D1 proteolysis, like its synthesis and assembly, is subject to mitogen regulation.

The subcellular distribution of D-type cyclins is also likely to be regulated by cell cycle-dependent events. Cyclin D1 accumulates in the nuclei of cells during G1 phase, but once DNA replication begins, it disappears from the nucleus (Baldin et al. 1993), despite the fact that its level of synthesis does not decrease markedly during S phase (Matsushime et al. 1991). The mechanisms that regulate the periodic subcellular redistribution of cyclin D1 during the cell division cycle have also not been defined.

We now demonstrate that glycogen synthase kinase-3β (GSK-3β) catalyzes the phosphorylation of cyclin D1 on Thr-286, thereby regulating cyclin D1 turnover in response to mitogenic signals. In turn, GSK-3β-mediated phosphorylation of cyclin D1 redirects the protein from the nucleus to the cytoplasm. Our results support a model in which phosphorylation of cyclin D1 on Thr-286 by GSK-3β links processes governing cyclin D1 subcellular localization with its proteasomal degradation.

/pdf/glycogen-synthase-kinase-3b-regulates-cyclin-d1-proteolysis-yriv13q4t5.pdf

Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization

GTP and isoproterenol activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in washed membranes prepared from C6 gliomas cells was enhanced by incubation with islet-activating protein, one of the pertussis toxins, if the incubation mixture was supplemented with NAD and ATP. The action of the protein was observed immediately after its addition and increased progressively in magnitude as the protein concentration or the incubation time increased. There was simultaneous incorporation of radioactivity from the ADP-ribose moiety of variously labeled NAD into the membrane protein with a molecular weight of 41,000. We conclude that islet-activating protein enhances receptor-mediated GTP-induced activation of membrane adenylate cyclase as a result of ADP-ribosylation of a membrane protein, probably one of the components of the receptor-adenylate cyclase system.

https://www.pnas.org/content/pnas/79/10/3129.full.pdf

Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein.

Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase

The subunit structure of islet-activating protein (IAP), pertussis toxin, has been analyzed to study a possibility that this protein is one of the A-B toxins [Gill, D. M. (1978) in Bacterial Toxins and Cell Membranes (Jeljaszewicz, J., & Wadstrom, T., Eds.) pp 291-332, Academic Press, New York]. Heating IAP with 1% sodium dodecyl sulfate caused its dissociation into five dissimilar subunits named S-1 (with a molecular weight of 28 000), S-2 (23 000), S-3 (22 000), S-4 (11 700), and S-5 (9300), as revealed by polyacrylamide gel electrophoresis; their molar ratio in the native IAP was 1:1:1:2:1. The molecular weight of IAP estimated by equilibrium ultracentrifugation was 117 000 which was not at variance with the value obtained by summing up molecular weights of the constituent subunits. The preparative separation of these IAP subunits was next undertaken; exposure of IAP to 5 M ice-cold urea for 4 days followed by column chromatography with carboxymethyl-Sepharose caused sharp separation of S-1 and S-5, leaving the other subunits as two dimers. These dimers were then dissociated into their constituent subunits, i.e., S-2 and S-4 for one dimer and S-3 and S-4 for the other, after 16-h exposure to 8 M urea; these subunits were obtained individually upon further chromatography on a diethylaminoethyl-Sepharose column. Subunits other than S-1 were adsorbed as a pentamer by a column using haptoglobin as an affinity adsorbent. The same pentamer was obtained by adding S-5 to the mixture of two dimers. Neither this pentamer nor other oligomers (or protomers) exhibited biological activity in vivo. Recombination of S-1 with the pentamer at the 1:1 molar ratio yielded a hexamer which was identical with the native IAP in electrophoretic mobility and biological activity to enhance glucose-induced insulin secretion when injected into rats. In the broken-cell preparation, S-1 was biologically as effective as the native IAP; both catalyzed ADP-ribosylation of a protein in membrane preparations from rat C6 glioma cells. In conclusion, IAP is an oligomeric protein consisting of an A (active) protomer (the biggest subunit) and a B (binding) oligomer which is produced by connecting two dimers by the smallest subunit in a noncovalent manner. Rationale for this terminology is discussed based on the A-B model.

Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model.

We have cloned cDNAs encoding alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go and determined their nucleotide sequences Purified preparations of Gi and Go alpha subunits (Gi alpha and Go alpha) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of Go alpha was obtained Then, the library was rescreened with a Go alpha cDNA probe to isolate several strongly or weakly hybridizing clones cDNAs encoding the complete sequences of Gi alpha and Gs alpha were thus obtained From nucleotide sequence analysis, the amino acid sequences of Gs alpha and Gi alpha were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively The calculated molecular weights for Gs alpha and Gi alpha were 45,663 and 40,499, respectively The Go alpha clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus The homology of the alpha subunits of Gs, Gi, Go, transducin, and ras-encoded protein is discussed

Michio Ui

Papers

Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein.

Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase

Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model.

Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.

A simple structure encodes G protein-activating function of the IGF-II/mannose 6-phosphate receptor