Stabilization of the GDP-bound conformation of Gialpha by a peptide derived from the G-protein regulatory motif of AGS3

The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein alpha subunits. To define the properties of this approximately 20-amino acid motif, we designed a GPR consensus peptide and determined its influence on the activation state of G-protein and receptor coupling to G-protein. The GPR peptide sequence (28 amino acids) encompassed the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevented the binding of AGS3 to Gialpha1,2 in protein interaction assays, inhibited guanosine 5'-O-(3-thiotriphosphate) binding to Gialpha, and stabilized the GDP-bound conformation of Gialpha. The GPR peptide had little effect on nucleotide binding to Goalpha and brain G-protein indicating selective regulation of Gialpha. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Gialpha. The GPR consensus peptide also blocked receptor coupling to Gialphabetagamma indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Gialpha, this conformation of Gialpha(GDP) was not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Gialpha- and Gbetagamma-regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems.     

Thermodynamic characterization of the binding of activator of G protein signaling 3 (AGS3) and peptides derived from AGS3 with G alpha i1

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) that contains four G protein regulatory (GPR) or GoLoco motifs in its C-terminal domain. The entire C-terminal domain (AGS3-C) as well as certain peptides corresponding to individual GPR motifs of AGS3 bound to G alpha i1 and inhibited the binding of GTP by stabilizing the GDP-bound conformation of G alpha i1. The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to G alpha i1 were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (Kd approximately 20 nm) and two apparent low affinity (Kd approximately 300 nm) binding sites for G alpha i1. Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (Kd approximately 400 nm) for G alpha i1. Peptides corresponding to each of the four GPR motifs of AGS3 (referred to as GPR1, GPR2, GPR3, and GPR4, respectively, going from N to C terminus) bound to G alpha i1 with Kd values in the range of 1-8 microm. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5'-3-O-(thio)triphosphate to G alpha i1, GPR3 did not. However, addition of N- and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for G alpha i1 and conferred GDI activity similar to that of AGS3-C itself. Similar increases were observed for extended GPR2 and extended GPR1 peptides. Thus, while the tertiary structure of AGS3 may affect the affinity and activity of the GPR motifs contained within its sequence, residues outside of the GPR motifs strongly potentiate their binding and GDI activity toward G alpha i1 even though the amino acid sequences of these residues are not conserved among the GPR repeats.     

Sequence analysis of cell cycle control (cdc2) protein kinases among protein serine/threonine kinases

Among protein serine/threonine kinases, the CDC2 proteins are both well characterized as protein serine/threonine kinases and are functionally involved in the control of cell division. Protein serine/threonine kinase sequences were analysed using Fourier transform of the coded sequences. Characteristic code/frequency pairs were extracted from a set of well defined protein serine/threonine kinases. The characteristic frequencies 0.179, 0.250 and 0.408 distinguished protein serine/threonine kinases from proteins which did not have the biological activity. Pertinent patterns in the sequence, responsible for the code/frequency pairs detection were searched and found to be correlated with the putative catalytic domain of the proteins. Protein serine/threonine kinases involved in cell division control, CDC2 protein kinases, were compared to the other protein serine/threonine kinases. Specific code/frequency pairs were extracted from the sequences and could be related to the function or regulation of the kinases in cell division. Two CDC2 related proteins CDC2(Mm) from mice and CDC2(Gg) from chicken were shown to fit well with the CDC2 proteins, whereas KIN28, PHO85 and PSKJ3, which share sequence homology but not functional activity with the CDC2 proteins, were clearly excluded from the CDC2 proteins by the characteristic code/frequency pairs. Pertinent patterns in the CDC2 proteins were analysed and mapped on the CDC2 related protein sequences. Four patterns were correlated with the code/frequency detection and therefore, could be associated to the regulation of the CDC2-related proteins.     

Identification of a human Akt3 (protein kinase B gamma) which contains the regulatory serine phosphorylation site

The family of protein kinases called Akt, protein kinase B (PKB), or related to A and C kinase (RAC) have been implicated in numerous biological processes including adipocyte and muscle differentiation, glycogen synthesis, glucose uptake, apoptosis and cellular proliferation. There are 3 known isoforms of this enzyme in mammalian cells (1/alpha, 2/beta and 3/gamma). Akt1 and 2 contain a key regulatory serine phosphorylation site in the carboxy-terminal region of the protein. However, the reported sequence of the rat Akt3 protein differed significantly from this in that it lacked 25 amino acids in the C-terminal region, including this key regulatory serine phosphorylation site (Biochem. Biophys. Res. Commun. 216, 526-534). In the present studies we show that the deduced sequence of human Akt3 contains this serine and that it is phosphorylated in response to insulin. These results indicate that human Akt3 is regulated similarly to Akt1 and Akt2.     

Positive and negative regulatory role of insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) serine/threonine phosphorylation

Insulin receptor substrates (IRS) 1 and 2 are phosphorylated on serine/threonine (Ser/Thr) residues in quiescent cells (basal phosphorylation), and phosphorylation on both Ser/Thr and tyrosine residues is increased upon insulin stimulation. To determine whether basal Ser/Thr phosphorylation of IRS proteins influences insulin receptor catalyzed tyrosine phosphorylation, recombinant FLAG epitope-tagged IRS-1 (F-IRS-1) and IRS-2 (F-IRS-2) were expressed, purified, and subjected to both dephosphorylation and hyperphosphorylation prior to phosphorylation by the insulin receptor kinase. As expected, hyperphosphorylation of F-IRS-1 and F-IRS-2 by GSK3beta decreased their subsequent phosphorylation on tyrosine residues by the insulin receptor. Surprisingly, however, dephosphorylation of the basal Ser/Thr phosphorylation sites impaired subsequent phosphorylation on tyrosine, suggesting that basal Ser/Thr phosphorylation of F-IRS-1 and F-IRS-2 plays a positive role in phosphorylation by the insulin receptor tyrosine kinase. Dephosphorylation of basal Ser/Thr sites on F-IRS-1 also significantly reduced tyrosine phosphorylation by the IGF-1 receptor. However, dephosphorylation of F-IRS-2 significantly increased phosphorylation by the IGF-1 receptor, suggesting that basal phosphorylation of IRS-2 has divergent effects on its interaction with the insulin and IGF-1 receptors. Phosphorylation of endogenous IRS-1 and IRS-2 from 3T3-L1 adipocytes was modulated in a similar manner. IRS-1 and IRS-2 from serum-fed cells were hyperphosphorylated, and dephosphorylation induced either by serum deprivation or by alkaline phosphatase treatment after immunoprecipitation led to an increase in tyrosine phosphorylation by the insulin receptor. Dephosphorylation of IRS-1 and IRS-2 immunoprecipitated from serum-deprived cells, however, resulted in inhibition of tyrosine phosphorylation by the insulin receptor. These data suggest that Ser/Thr phosphorylation can have both a positive and a negative regulatory role on tyrosine phosphorylation of IRS-1 and IRS-2 by insulin and IGF-1 receptors.     

Human immunodeficiency virus type 1 Vpr-mediated G(2) cell cycle arrest: Vpr interferes with cell cycle signaling cascades by interacting with the B subunit of serine/threonine protein phosphatase 2A

The Vpr protein of primate lentiviruses arrests cell cycling at the G(2)/M phase through an inactivation of cyclin B-p34(cdc2) and its upstream regulator cdc25. We provide here biochemical and functional evidence demonstrating that human immunodeficiency virus type 1 (HIV-1) Vpr mediates G(2) arrest by forming a complex with protein phosphatase 2A (PP2A), an upstream regulator of cdc25. Vpr associates with PP2A through a specific interaction with the B55 regulatory subunit. This interaction is necessary but not sufficient for G(2) arrest. Interestingly, we found that Vpr association with B55-containing PP2A targets the enzymatic complex to the nucleus and, importantly, enhances the recruitment and dephosphorylation of the cdc25 substrate. Our data suggest that Vpr mediates G(2) arrest by enhancing the nuclear import of PP2A and by positively modulating its catalytic activity towards active phosphorylated nuclear cdc25.     

Expression analysis of a novel p75(NTR) signaling protein,which regulates cell cycle progression and apoptosis

Neurotrophin receptor-interacting MAGE (NRAGE) is the most recently identified p75 neurotrophin receptor (p75(NTR)) intracellular binding protein. Previously, NRAGE over-expression was shown to mediate cell cycle arrest and facilitate nerve growth factor (NGF) dependent apoptosis of sympathetic neuroblasts in a p75(NTR) specific manner. Here we have examined the temporal and spatial expression patterns of NRAGE over the course of murine embryogenesis to determine whether NRAGE's expression is consistent with its proposed functions. We demonstrate that NRAGE mRNA and protein are expressed throughout embryonic and adult tissues. The mRNA is constitutively expressed within each tissue across development. However, expression of NRAGE protein displays a tight temporal tissue specific regulation. During early CNS development, NRAGE protein is expressed throughout the neural tube, but by later stages of neurogenesis, NRAGE protein is restricted within the ventricular zone, subplate and cortical plate. Moreover, NRAGE protein expression is limited to proliferative neural subpopulations as we fail to detect NRAGE expression co-localized with mature/differentiation associated neuronal markers. Interestingly, NRAGE's expression is not restricted solely to areas of p75(NTR) expression suggesting that NRAGE may mediate proliferation and/or apoptosis from other environmental signals in addition to NGF within the CNS. Our data support previously characterized roles for NRAGE as a mediator of precursor apoptosis and a repressor of cell cycle progression in neural development.     

Altered signaling and cell cycle regulation in embryonal stem cells with a disruption of the gene for phosphoinositide 3-kinase regulatory subunit p85alpha

The p85alpha regulatory subunit of class I(A) phosphoinositide 3-kinases (PI3K) is derived from the Pik3r1 gene, which also yields alternatively spliced variants p50alpha and p55alpha. It has been proposed that excess monomeric p85 competes with functional PI3K p85-p110 heterodimers. We examined embryonic stem (ES) cells with heterozygous and homozygous disruptions in the Pik3r gene and found that wild type ES cells express virtually no monomeric p85alpha. Although, IGF-1-stimulated PI3K activity associated with insulin receptor substrates was unaltered in all cell lines, p85alpha-null ES cells showed diminished protein kinase B activation despite increased PI3K activity associated with the p85beta subunit. Furthermore, p85alpha-null cells demonstrated growth retardation, increased frequency of apoptosis, and altered cell cycle regulation with a G(0)/G(1) cell cycle arrest and up-regulation of p27(KIP), whereas signaling through CREB and MAPK was enhanced. These phenotypes were reversed by re-expression of p85alpha via adenoviral gene transfer. Surprisingly, all ES cell lines could be differentiated into adipocytes. In these differentiated ES cells, however, compensatory p85beta signaling was lost in p85alpha-null cells while increased signaling by CREB and MAPK was still observed. Thus, loss of p85alpha in ES cells induced alterations in IGF-1 signaling and regulation of apoptosis and cell cycle but no defects in differentiation. However, differentiated ES cells partially lost their ability for compensatory signaling at the level of PI3K, which may explain some of the defects observed in mice with homozygous deletion of the Pik3r1 gene.     

6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27

The control of glucose metabolism and the cell cycle must be coordinated in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are well established regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent allosteric activator of 6-phosphofructo-1-kinase (Pfk-1). PFKFB3 is overexpressed in human cancers, regulated by HIF-1α, Akt and PTEN, and required for the survival and growth of multiple cancer types. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, activates cyclin-dependent kinases (Cdks). In particular, F2,6BP stimulates the Cdk-mediated phosphorylation of the Cip/Kip protein p27 (threonine 187), which in turn results in p27''s ubiquitination and proteasomal degradation. As p27 is a potent suppressor of the G1/S transition and activator of apoptosis, we hypothesized that the known requirement of PFKFB3 for cell cycle progression and prevention of apoptosis may be partly due to the ability of F2,6BP to activate Cdks. In this study, we demonstrate that siRNA silencing of endogenous PFKFB3 inhibits Cdk1 activity, which in turn stabilizes p27 protein levels causing cell cycle arrest at G1/S and increased apoptosis in HeLa cells. Importantly, we demonstrate that the increase in apoptosis and suppression of the G1/S transition caused by siRNA silencing of PFKFB3 expression is reversed by co-siRNA silencing of p27. Taken together with prior publications, these observations support a model whereby PFKFB3 and F2,6BP function not only as regulators of Pfk-1 but also of Cdk1 activity, and therefore serve to couple glucose metabolism with cell proliferation and survival in transformed cells.The homodimeric bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB) phosphorylate fructose 6-phosphate (F6P) to fructose-2,6-bisphosphate (F2,6BP), which in turn activates 6-phosphofructo-1-kinase and glycolytic flux to lactate.¹ Of the four genes encoding distinct PFKFB isozymes (PFKFB1-4), PFKFB3 is distinguished by the presence of multiple copies of the AUUUA instability motif in its 3''untranslated region,² a very high kinase:phosphatase activity ratio (740 : 1),³ increased protein expression in rapidly proliferating transformed cells,² solid tumors and leukemias^{2, 4, 5} and regulation by several proteins essential for tumor progression (e.g. HIF-1α,⁶ Akt⁷ and PTEN^{8, 9}). Not surprisingly, heterozygous genomic deletion of the pfkfb3 gene has been found to reduce both the glucose metabolism and growth of Ras-transformed tumors in syngeneic mice.¹⁰In recent studies, we unexpectedly observed that PFKFB3 trafficked to the nucleus of multiple cell lines via a highly conserved nuclear localization motif in the C-terminal domain.¹¹ Although the precise role of nuclear PFKFB3 is unknown, ectopic expression of wild-type PFKFB3 in the nucleus was found to stimulate cellular proliferation without affecting glycolysis, suggesting a novel role for nuclear F2,6BP in regulating the cell cycle.¹¹ Moreover, the addition of F2,6BP to total cell lysates was found to increase the cyclin-dependent kinase (Cdk)-dependent phosphorylation of its substrate p27 at threonine 187 (T187), a posttranslational modification that targets p27 for degradation (i.e. high Cdk activity suppresses p27 levels).¹¹ Given that p27 can potently block the G1/S transition and stimulate apoptosis, these data indicated that PFKFB3-mediated production of F2,6BP in the nucleus may directly stimulate Cdks to phosphorylate T187-p27, targeting p27 for degradation by the proteasome and allowing cells to both proliferate and evade apoptosis. Furthermore, these data signified that PFKFB3 may not only be essential for the regulation of glycolysis in the cytoplasm but also for the control of the cell cycle in the nucleus.Based on these prior studies, we postulated that selective inhibition of PFKFB3 would suppress Cdk1 activity, which in turn would reduce the phosphorylation of T187-p27, resulting in increased p27 expression, reduced G1/S transition and increased apoptosis. We provide evidence to support this chain of biochemical and cellular events after PFKFB3 inhibition as well as direct verification that p27 itself is required for the simultaneous suppression of G1/S transition and induction of apoptosis caused by PFKFB3 inhibition. Given that PFKFB3 inhibitors are entering phase I trials for the treatment of advanced cancers,¹² we believe that this new mechanism of action may facilitate the development of rational phase I/II trials that combine other apoptosis-activating agents that disrupt p27 function (e.g. Cdk1 inhibitors) as well as potential biomarkers such as p27 that may demonstrate the on-target effects of PFKFB3 inhibitors in biopsies and resected tumors. From a broader perspective, these data provide further support for the concept that PFKFB3 may be an essential coupler of glucose metabolism and cell cycle progression.     

Yeast Rio1p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression

Rio1p was identified as a protein serine kinase founding a novel subfamily. It is highly conserved from Archaea to man and only distantly related to previously established protein kinase families. Nevertheless, analysis of multiple protein sequence alignments shows that those amino acid residues that are important for either structure or catalytic activity in conventional protein kinases are also conserved in members of the Rio1p family at the respective positions (corresponding to domains I-XI of protein kinases). Recombinant Rio1p from Escherichia coli and tagged Rio1p from yeast has kinase activity in vitro, and mutation of amino acid residues that are conserved and indispensable for catalytic activity (i.e. ATP-binding motif, catalytic centre) abrogates activity. RIO1 is essential in yeast and plays a role in cell cycle progression. After sporulation of RIO1/rio1 diploids, RIO1-disrupted progeny cease growth after one to three cell divisions and arrest as either large unbudded or large-budded cells. Cells deprived of Rio1p are enlarged and arrest either in G1 or in mitosis mainly with the DNA at the bud neck and short spindles (a phenotype also seen in cells carrying a weak allele), suggesting that Rio1p activity is required for at least at two steps during the cell division cycle: for entrance into S phase and for exit from mitosis. The weak RIO1 allele leads to increased plasmid loss.     

Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21

Replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in various processes, including nucleotide excision repair and DNA replication. The 32 kDa subunit of RPA (RPA32) is phosphorylated in response to various DNA-damaging agents, and two protein kinases, ataxia-telangiectasia mutated (ATM) and the DNA-dependent protein kinase (DNA-PK) have been implicated in DNA damage-induced phosphorylation of RPA32. However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported. Here we generated a phosphospecific antibody that recognizes Thr²¹-phosphorylated RPA32. We show that both DNA-PK and ATM phosphorylate RPA32 on Thr²¹ in vitro. Ionizing radiation (IR)-induced phosphorylation of RPA32 on Thr²¹ was defective in ATM-deficient cells, while camptothecin (CPT)-induced phosphorylation of RPA32 on Thr²¹ was defective in cells lacking functional DNA-PK. Neither ATM nor DNA-PK was required for etoposide (ETOP)-induced RPA32 Thr²¹ phosphorylation. However, two inhibitors of the ATM- and Rad3-related (ATR) protein kinase activity prevented ETOP-induced Thr²¹ phosphorylation. Inhibition of DNA replication prevented both the IR- and CPT-induced phosphorylation of Thr²¹, whereas ETOP-induced Thr²¹ phosphorylation did not require active DNA replication. Thus, the regulation of RPA32 Thr²¹ phosphorylation by multiple DNA damage response protein kinases suggests that Thr²¹ phosphorylation of RPA32 is a crucial step within the DNA damage response.     

G-protein alpha subunit interaction and guanine nucleotide dissociation inhibitor activity of the dual GoLoco motif protein PCP-2 (Purkinje cell protein-2)

Purkinje cell protein-2 (PCP-2; L7/GPSM4) is a GoLoco motif-containing protein that is specifically expressed in Purkinje and retinal ON bipolar cells. An alternative splice variant of PCP-2 has recently been isolated which contains two GoLoco motifs. Although the second GoLoco motif (GL2) of PCP-2 has been reported to interact with Galpha-subunits, a complete biochemical analysis of each individual motif of PCP-2 has not been performed. We demonstrate that the first GoLoco motif (GL1) of PCP-2 is equipotent as a guanine nucleotide dissociation inhibitor (GDI) towards Galphai1 and Galphai2, while it has sevenfold lower GDI activity for Galphai3 and greater than 20-fold lower GDI activity against Galphao. In contrast we found PCP-2 GL2 to be essentially equipotent as a GDI for all Galphai subunits, but it had negligible activity toward Galphao. Using co-immunoprecipitation from COS-7 cells, we found that PCP-2 was only able to interact with Galphai1 but not Galphao nor Galpha-subunits from other families (Galphas, Galphaq, or Galpha12). Mutational analysis of a non-canonical residue (glycine 24) in human PCP-2 GL1 provided evidence for heterogeneity in mechanisms of Galphai interactions with GoLoco motifs. Collectively, the data demonstrate that PCP-2 is a comparatively weak GoLoco motif protein that exhibits highest affinity interactions and GDI activity toward Galphai1, Galphai2, and Galphai3 subunits.     

The G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and Gialpha3 influence cortical positioning of the mitotic spindle poles at metaphase in symmetrically dividing mammalian cells

We addressed the role of the G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and G-proteins (Gialpha3) in the positioning of the spindle pole during mammalian cell division. Immunocytochemistry indicated that both LGN and Gialpha3 co-localized at the spindle pole and at the midbody and the cell cortex during the different phases of mitosis. In marked contrast to the positioning of the spindle pole at metaphase midway between the cell cortex and the metaphase plate, the spindle pole was juxtaposed with the cell cortex at metaphase following increased expression of Gialpha3 and LGN. This repositioning of the spindle pole required the interaction of LGN with Gialpha. The influence of LGN and Gialpha3 on the cortical positioning of the spindle pole likely reflects either stronger pulling forces on the spindle pole exerted from the cell cortex or increased pushing forces exerted on the spindle pole from the mitotic spindle indicating that these events are regulated by GPR motif-containing proteins and G-proteins independent of asymmetry.     

Feedback inhibition on cell wall integrity signaling by Zds1 involves Gsk3 phosphorylation of a cAMP-dependent protein kinase regulatory subunit

We report here that budding yeast cAMP-dependent protein kinase (cAPK) is controlled by heat stress. A rise in temperature from 30 to 37 degrees C was found to result in both a higher expression and an increased cytoplasmic localization of its regulatory subunit Bcy1. Both of these effects required phosphorylation of serines located in its localization domain. Surprisingly, classic cAPK-controlled processes were found to be independent of Bcy1 phosphorylation, indicating that these modifications do not affect cAPK activity as such. Alternatively, phosphorylation may recruit cAPK to, and thereby control, a specific subset of (perhaps novel) cAPK targets that are presumably localized extranuclearly. Zds1 and Zds2 may play a role in this process, since these were found required to retain hyperphosphorylated Bcy1 in the cytoplasm at 37 degrees C. Mck1, a homologue of mammalian glycogen synthase kinase 3 and a downstream component of the heat-activated Pkc1-Slt2/Mpk1 cell wall integrity pathway, is partly responsible for hyperphosphorylations of Bcy1. Remarkably, Zds1 appears to act as a negative regulator of cell wall integrity signaling, and this activity is dependent in part on the phosphorylation status of Bcy1. Thus, Mck1 phosphorylation of Bcy1 and Zds1 may constitute an unprecedented negative feedback control on the cell wall integrity-signaling pathway.