C-terminal Phosphorylation Sites Research Articles

The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation

The positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26 degrees of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis.

ATPase activity and its temperature compensation of the cyanobacterial clock protein KaiC

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria. KaiC is a homohexamer; its subunit contains duplicated halves, each with a set of ATPase motifs. Here, using highly purified KaiC preparations of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 produced in Escherichia coli, we found that the N- and C-terminal domains of KaiC had extremely weak ATPase activity. ATPase activity showed temperature compensation in wild-type KaiC, but not in KaiC(S431A/T432A), a mutant that lacks two phosphorylation sites. We concluded that KaiC phosphorylation is involved in the ATPase temperature-compensation mechanism-which is probably critical to the stability of the circadian clock in cyanobacteria-and we hypothesized the following temperature-compensation mechanism: (i) The C-terminal phosphorylation sites of a KaiC hexamer subunit are phosphorylated by the C-terminal domain of an adjacent KaiC subunit; (ii) the phosphorylation suppresses the ATPase activity of the C-terminal domain; and (iii) the phosphorylated KaiC spontaneously dephosphorylates, resulting in the recover of ATPase activity.

A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P(2) (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P(2) in regulating glucose metabolism and insulin secretion in pancreatic beta-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P(2), in regulating beta-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P(2) were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P(2) content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P(2) in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P(2), in potentiating glucose-induced insulin secretion. Whereas down-regulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P(2) is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

Sae2p phosphorylation is crucial for cooperation with Mre11p for resection of DNA double-strand break ends during meiotic recombination in Saccharomyces cerevisiae

Meiotic recombination is initiated by the introduction of DNA double-strand breaks (DSBs) at recombination hotspots. DSB ends are resected to yield ssDNA, which is used in a homology search. Sae2p, which is involved in the resection of DSB ends, is phosphorylated by the Mec1p and Tel1p kinases during meiosis. To clarify the role of Sae2p phosphorylation in meiotic recombination, three mutants with alanine substitutions (at two putative Mec1/Tel1 phosphorylation sites near the N terminus, at three sites near the C terminus or at all five sites) were constructed. Analysis of DSB ends during meiotic recombination demonstrated that phosphorylation of the three C-terminal phosphorylation sites is necessary for DSB end resection and that phosphorylation of the two N-terminal phosphorylation sites is required for the efficient initiation of DSB end resection. Sae2p was localized on meiotic chromosomes in the rad50S and mre11-H125R mutants, which accumulate DSB ends. Alanine substitutions of all phosphorylation sites did not affect localization of Sae2p on meiotic chromosomes. Although colocalization of Sae2p with Mre11p and recombinant formation were observed in the N-terminally mutated and the C-terminally mutated strains, these processes were drastically impaired in the quintuple mutant. These results indicate that phosphorylation of Sae2p is required to initiate resection and to improve the efficiency of resection through cooperation with the Mre11-Rad50-Xrs2 complex.

Trafficking-dependent phosphorylation of Kv1.2 regulates voltage-gated potassium channel cell surface expression

Kv1.2 alpha-subunits are components of low-threshold, rapidly activating voltage-gated potassium (Kv) channels in mammalian neurons. Expression and localization of Kv channels is regulated by trafficking signals encoded in their primary structure. Kv1.2 is unique in lacking strong trafficking signals and in exhibiting dramatic cell-specific differences in trafficking, which is suggestive of conditional trafficking signals. Here we show that a cluster of cytoplasmic C-terminal phosphorylation sites regulates Kv1.2 trafficking. Using tandem MS to analyze Kv1.2 purified from rat, human, and mouse brain, we identified in each sample in vivo phosphoserine (pS) phosphorylation sites at pS434, pS440, and pS441, as well as doubly phosphorylated pS440/pS441. We also found these sites, as well as pS449, on recombinant Kv1.2 expressed in heterologous cells. We found that phosphorylation at pS440/pS441 is present only on the post-endoplasmic reticulum (ER)/cell surface pool of Kv1.2 and is not detectable on newly synthesized and ER-localized Kv1.2, on which we did observe pS449 phosphorylation. Elimination of PS440/PS441 phosphorylation by mutation reduces cell-surface expression efficiency and functional expression of homomeric Kv1.2 channels. Interestingly, mutation of S449 reduces phosphorylation at pS440/pS441 and also decreases Kv1.2 cell-surface expression efficiency and functional expression. These mutations also suppress trafficking of Kv1.2/Kv1.4 heteromeric channels, suggesting that incorporation of Kv1.2 into heteromeric complexes confers conditional phosphorylation-dependent trafficking to diverse Kv channel complexes. These data support Kv1.2 phosphorylation at these clustered C-terminal sites as playing an important role in regulating trafficking of Kv1.2-containing Kv channels.

Temporal Analysis of Sucrose-induced Phosphorylation Changes in Plasma Membrane Proteins of Arabidopsis

Sucrose is the main product of photosynthesis and the most common transport form of carbon in plants. In addition, sucrose is a compound that serves as a signal affecting metabolic flux and development. Here we provide first results of externally induced phosphorylation changes of plasma membrane proteins in Arabidopsis. In an unbiased approach, seedlings were grown in liquid medium with sucrose and then depleted of carbon before sucrose was resupplied. Plasma membranes were purified, and phosphopeptides were enriched and subsequently analyzed quantitatively by mass spectrometry. In total, 67 phosphopeptides were identified, most of which were quantified over five time points of sucrose resupply. Among the identified phosphorylation sites, the well described phosphorylation site at the C terminus of plasma membrane H(+)-ATPases showed a relative increase in phosphorylation level in response to sucrose. This corresponded to a significant increase of proton pumping activity of plasma membrane vesicles from sucrose-supplied seedlings. A new phosphorylation site was identified in the plasma membrane H(+)-ATPase AHA1 and/or AHA2. This phosphorylation site was shown to be crucial for ATPase activity and overrode regulation via the well known C-terminal phosphorylation site. Novel phosphorylation sites were identified for both receptor kinases and cytosolic kinases that showed rapid increases in relative intensities after short times of sucrose treatment. Seven response classes were identified including non-responsive, rapid increase (within 3 min), slow increase, and rapid decrease. Relative quantification of phosphorylation changes by phosphoproteomics provides a means for identification of fast responses to external stimuli in plants as a basis for further functional characterization.

MGlu4 potentiation of K 2P2.1 is dependant on C-terminal dephosphorylation

Two-pore domain potassium (K 2P) channels are proposed to underlie the background or leak current found in many excitable cells. Extensive studies have been performed investigating the inhibition of K 2P2.1 by Gα q- and Gα s-coupled G-protein-coupled receptors (GPCRs), whereas in the present study we investigate the mechanisms underlying Gα i/Gα o-coupled GPCR increases in K 2P2.1 activity. Activation of mGlu4 increases K 2P2.1 activity, with pharmacological inhibition of protein kinases and phosphatases revealing the involvement of PKA whereas PKC, PKG or protein phosphatases play no role. Mutational analysis of potential C-terminal phosphorylation sites indicates S333 to control approximately 70%, with S300 controlling approximately 30% of the increase in K 2P2.1 activity following mGlu4 activation. These data reveal that activation of mGlu4 leads to an increase in K 2P2.1 activity through a reduction in C-terminal phosphorylation, which represents a novel mechanism by which group III mGlu receptors may regulate cell excitability and synaptic activity.

Structural Basis for Spinophilin−Neurabin Receptor Interaction

Neurabin and spinophilin are neuronal scaffolding proteins that play important roles in the regulation of synaptic transmission through their ability to target protein phosphatase 1 (PP1) to dendritic spines where PP1 dephosphorylates and inactivates glutamate receptors. However, thus far, it is still unknown how neurabin and spinophilin themselves are targeted to these membrane receptors. Spinophilin and neurabin contain a single PDZ domain, a common protein-protein interaction recognition motif, which are 86% identical in sequence. We report the structures of both the neurabin and spinophilin PDZ domains determined using biomolecular NMR spectroscopy. These proteins form the canonical PDZ domain fold. However, despite their high degree of sequence identity, there are distinct and significant structural differences between them, especially between the peptide binding pockets. Using two-dimensional 1H-15N HSQC NMR analysis, we demonstrate that C-terminal peptide ligands derived from glutamatergic AMPA and NMDA receptors and cytosolic proteins directly and differentially bind spinophilin and neurabin PDZ domains. This peptide binding data also allowed us to classify the neurabin and spinophilin PDZ domains as the first identified neuronal hybrid class V PDZ domains, which are capable of binding both class I and II peptides. Finally, the ability to bind to glutamate receptor subunits suggests that the PDZ domains of neurabin and spinophilin are important for targeting PP1 to C-terminal phosphorylation sites in AMPA and NMDA receptor subunits.

Dynamics of Protein Kinase C-mediated Phosphorylation of the Complement C5a Receptor on Serine 334

Upon agonist binding, the C5a anaphylatoxin receptor (C5aR) is rapidly phosphorylated on phosphorylation sites that are located within the C-terminal domain of the receptor. Previous studies suggested that C5aR phosphorylation proceeds in a hierarchical manner with serine 334 presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. To better understand the dynamics of Ser-334 phosphorylation, we generated site-specific monoclonal antibodies that specifically react with phosphoserine 334. In differentiated U937 cells, which endogenously express C5aR, stimulation with low C5a concentrations resulted in a very rapid (t((1/2)) approximately 20 s), albeit transient, receptor phosphorylation. Whole cell phosphorylation assays with specific inhibitors as well as in vitro phosphorylation assays with recombinant enzymes and peptide substrates revealed that phosphorylation of Ser-334 is regulated by protein kinase C-beta and a calyculin A-sensitive protein phosphatase. Surprisingly, at high concentrations (>10 nM) of C5a, the protein kinase C-mediated phosphorylation of Ser-334 was essentially blocked. This could be attributed to the even faster (t((1/2)) < 5 s) binding of beta-arrestin to the receptor. Analysis of C5aR Ser/Ala mutants that possess a single intact serine residue either at position 334 or at neighboring positions 327, 332, or 338 revealed functional redundancy of C-terminal phosphorylation sites since all 4 serine residues could individually support C5aR internalization and desensitization. This study is among the first to analyze in a detailed manner, using a non-mutational approach, modifications of a defined phosphorylation site in a G protein-coupled receptor and to correlate these findings with functional parameters of receptor deactivation.

Potentiation of Smad-mediated transcriptional activation by the RNA-binding protein RBPMS.

Smad2, Smad3 and Smad4 proteins are considered to be key mediators of transforming growth factor-β (TGF-β) signaling. However, the identities of the Smad partners mediating TGF-β signaling are not fully understood. Here, we show that RNA-binding protein with multiple splicing (RBPMS), a member of the RNA-binding protein family, physically interacts with Smad2, Smad3 and Smad4 both in vitro and in vivo. The presence of TGF-β increases the binding of RBPMS with these Smad proteins. Consistent with the binding results, overexpression of RBPMS enhances Smad-dependent transcriptional activity in a TGF-β-dependent manner, whereas knockdown of RBPMS decreases this activity. RBPMS interacts with TGF-β receptor type I (TβR-I), increases phosphorylation of C-terminal SSXS regions in Smad2 and Smad3, and promotes the nuclear accumulation of the Smad proteins. Moreover, RBPMS fails to enhance the transcriptional activity of Smad2 and Smad3 that lack the C-terminal phosphorylation sites. Our data provide the first evidence for an RNA-binding protein playing a role in regulation of Smad-mediated transcriptional activity and suggest that RBPMS stimulates Smad-mediated transactivation possibly through enhanced phosphorylation of Smad2 and Smad3 at the C-terminus and promotion of the nuclear accumulation of the Smad proteins.

C-terminal Src Kinase-homologous Kinase (CHK), a Unique Inhibitor Inactivating Multiple Active Conformations of Src Family Tyrosine Kinases

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.

Different endoplasmic reticulum trafficking and processing pathways for calsequestrin (CSQ) and epitope-tagged CSQ

Cardiac calsequestrin (CSQ) is a protein that traffics to and concentrates inside sarcoplasmic reticulum (SR) terminal cisternae, a protein secretory compartment of uncertain origin. To investigate trafficking of CSQ within standard ER compartments, we expressed CSQ in nonmuscle cell lines and examined its localization by immunofluorescence and its molecular structure from the mass spectrum of total cellular CSQ. In all cells examined, CSQ was a highly phosphorylated protein with a glycan structure predictive of ER-retained proteins: Man9,8GlcNAc2 lacking terminal GlcNAc. Immunostaining was restricted to polymeric ER cisternae. Secretory pathway disruption by brefeldin A and thapsigargin led to altered CSQ glycosylation and phosphorylation consistent with post-ER trafficking. When epitope-tagged forms of CSQ were expressed in the same cells, mannose trimming of CSQ glycans was far more extensive, and C-terminal phosphorylation sites were nearly devoid of phosphate, in complete contrast to the highly phosphorylated wild-type protein that concentrates in all cells tested. Epitope-tagged CSQ also showed a reduced ER staining compared to wild-type protein, with significant staining in juxta-Golgi compartments. Loss of ER retention due to epitope tags or thapsigargin and resultant changes in protein structure or levels of bound Ca 2+ point to CSQ polymerization as an ER/SR retention mechanism.

Phosphorylation of Ime2 Regulates Meiotic Progression in Saccharomyces cerevisiae

Ime2p is a meiosis-specific protein kinase in Saccharomyces cerevisiae that controls multiple steps in meiosis. Although Ime2p is functionally related to the Cdc28p cyclin-dependent kinase (CDK), no cyclin binding partners that regulate its activities have been identified. The sequence of the Ime2p catalytic domain is similar to CDKs and mitogen-activated protein kinases (MAPKs). Ime2p is activated by phosphorylation of its activation loop in a Cak1p-dependent fashion and is subsequently phosphorylated on multiple residues as cells progress through meiosis. In this study, we show that Ime2p purified from meiotic cells is phosphorylated on Thr(242) and Tyr(244) in its activation loop and on Ser(520) and Ser(625) in its C terminus. Ime2p autophosphorylates on threonine in its activation loop in vitro consistent with autophosphorylation of Thr(242) playing a role in its activation. Moreover, autophosphorylation in cis is required for Ime2p to become hyperphosphorylated. Phosphorylation of the C-terminal serines is not essential to sporulation. However, Ime2p C-terminal phosphorylation site mutants genetically interact with components of the FEAR network that controls exit from meiosis I. These data suggest that Ime2p plays a role in controlling the exit from meiosis I and demonstrate that a phospho-modification pathway regulates Ime2p during the different phases of meiotic development.

A novel leptin signalling pathway via PTEN inhibition in hypothalamic cell lines and pancreatic β-cells

In obesity and diabetes, the ability of hypothalamic neurons to sense and transduce changes in leptin and insulin levels is compromised. The effects of both hormones require intracellular signalling via the PI3-kinase pathway, which is inhibited by the phosphatase PTEN. We show that leptin-stimulated F-actin depolymerization in mouse hypothalamic cells is inhibited by PTEN, a process involving independent effects of both its lipid and protein phosphatase activities. Potentially mediating this F-actin depolymerization, leptin, but not insulin, stimulated the phosphorylation of PTEN in a CK2 dependent manner, and inhibited its phosphatase activity. Similarly, hyperpolarization of mouse pancreatic beta-cells by leptin also requires coincident PtdIns(3,4,5)P3 generation and actin depolymerization, and could be inhibited by mechanisms requiring both the lipid and protein phosphatase activities of PTEN. These results demonstrate a critical role for PTEN in leptin signalling and indicate a mechanism by which leptin and insulin can produce PI3K dependent differential cellular outputs.

CK2-dependent C-terminal phosphorylation at T 300 directs the nuclear transport of TSPY protein

TSPY (testis-specific protein, Y-encoded) is a member of the greater SET/NAP family of molecules with various functions, e.g., in chromatin remodeling, regulation of gene expression, and has been implicated to play a role in the malignant development of gonadoblastoma, testicular and prostate cancer. Here we demonstrate that the C-terminus has a functional role for the nucleo-cytoplasmatic shuttling of the TSPY protein. Using various combinations of in vitro mutagenesis and enhanced green fluorescent protein reporter gene-expression experiments we were able to show that while the deletion of C-terminus leads to a decreased stability and enhanced degradation of the protein, the selective mutation of a C-terminal CK2 phosphorylation site (T 300) prevents the TSPY protein from entering the nucleus. We conclude that phosphorylation of the (T 300) residue is a necessary and functional prerequisite for TSPY’s transport into the nucleus reminding of comparable data from a related Drosophila molecule, NAP1.

RUNX1/AML1 Is Phosphorylated at Both Its N- and C-Terminus by cdk6/cyclin D3 or cdk1/cyclin B.

RUNX1/AML1 is a key transcriptional mediator of hematopoiesis and leukemogenesis. AML1 regulates myeloid and lymphoid differentiation via activation of lineage-specific genes such as those encoding myeloperoxidase or the T cell receptor δ and participates in apoptotic response pathways via its ability to tranactivate the p14/p19ARF gene. In addition, AML1 accelerates G1 to S cell cycle progression, via activation of the cyclin D3 and potentially the cdk4 genes. CBF oncoproteins such as AML1-ETO or CBFβ-SMMHC interfere with the activities of AML1 and block myeloid differentiation and slow cell cycle progression, and mutations such as loss of p16 which accelerate G1 prevent cell cycle inhibition and cooperate with CBF oncoproteins to induce acute leukemia in mice. In addition to regulation of the cell cycle by AML1, we have been interested in how AML1 activities vary and may be regulated during cell cycle progression. We recently reported that endogenous AML1 levels increase in hematopoietic cell lines as they progress from G1 to S and then diminish again at the end of mitosis (Bernardin-Fried et al J. Biol. Chem. 279:15678, 2004). RNA levels did not vary, but exogenous AML1 mimicked the behaviour of the endogenous protein, suggesting regulation at the level of protein stability. Mutation of two Ras-dependent phosphorylation sites, S276 and S293, to alanine did not prevent cell cycle variation. We have therefore set out to evaluate whether AML1 stability might be regulated by cyclin-dependent kinase (cdk) phosphorylation. AML1 contains 480 amino acids and binds DNA via its N-terminal Runt domain. Both the cdk6/cyclin D3 and the cdk1/cyclin B complex, expressed from baculovirus vectors, phosphorylated GST-AML1(1-290) and GST-AML1(290–480). The Runt domain alone, in GST-AML1(86–217), was not phosphorylated. Interestingly, exogenous DNA-binding domain alone did not vary during the cell cycle. This is the first demonstration that a specific kinase phosphorylates AML1 in vitro. There are three (S/T)PX(K/R) cdk consensus sites in AML1, with serines at residues 48, 303, and 424. Mutation of S424 to alanine did not prevent phosphorylation of GST-AML1(290–480). Additional mutations of these and other serines or threonines adjacent to proline are being generated to further map the cdk phosphorylation sites and to enable in vivo experiments designed to evaluate the effects of these mutations on cell cycle-specific AML1 expression. We propose a model in which accumulated phosphorylation of AML1 during the S and G2/M cell cycle phases leads to ubiquitin-mediated AML1 destabilization at the end of mitosis. The increased stability of AML1 in the presence of proteosome inhibitors supports this model. Phosphorylation-mediated destabilization of AML1 may complement the recent finding that direct interaction of cyclin D3 with AML1 inhibits its activity as a transcriptional activator. Each of these mechanisms may help regulate the proliferation of hematopoietic stem/progenitor cells. Finally, perhaps loss of destabilizing C-terminal phosphorylation sites in the AML1-ETO oncoprotein increases its ability to dominantly repress AML1-target genes during myeloid leukemogenesis.

Role of the C Terminus in Neuropeptide Y Y Receptor Desensitization and Internalization

We have studied truncation mutants of the rat neuropeptide Y (NPY) Y1 receptor lacking four (Thr361stop, Y1T361*) or eight (Ser352stop, Y1S352*) potential serine/threonine C-terminal phosphorylation sites. NPY-stimulated hemagglutinin-tagged Y1, Y1T361*, and Y1S352* receptors all efficiently activated G proteins in Chinese hamster ovary (CHO) cell membranes, but desensitization after NPY pretreatment was only prevented in the HAY1S352* clone. In transfected colonic carcinoma epithelial layers, functional Y1 and Y1T361* peptide YY responses became more transient as the agonist concentration increased, whereas those mediated by the Y1S352* receptor remained sustained. NPY-stimulated HAY1 receptor phosphorylation was increased by transient overexpression of G protein-coupled receptor kinase 2, and only Ser352stop truncation abolished this response in CHO or human embryonic kidney (HEK) 293 cells. Rapid internalization of cell-surface HAY1 receptors in HEK293 cells was observed in response to agonist, resulting in partial colocalization with transferrin, a marker for clathrin-mediated endocytosis and recycling. It is surprising that both truncated receptors were constitutively internalized, predominantly in transferrin-positive compartments. NPY increased cell-surface localization of HAY1S352* receptors, whereas the distribution of both mutants was unaltered by BIBO3304. Recruitment of green fluorescent protein-tagged beta-arrestin2 to punctate endosomes was observed only for HAY1 and HAY1T361* receptors and solely under NPY-stimulated conditions. Thus, the key C-terminal sequence between Ser352 and Lys360 is a major site for Y1 receptor phosphorylation, is critical for its desensitization, and contributes to the association between the receptor and beta-arrestin proteins. However, additional beta-arrestin-independent mechanisms control Y1 receptor trafficking under basal conditions.

PVR plays a critical role via JNK activation in thorax closure during Drosophila metamorphosis.

PVR, the Drosophila homolog of the PDGF/VEGF receptor, has been implicated in border cell migration during oogenesis and hemocyte migration during embryogenesis. It was earlier shown that Mbc, a CDM family protein, and its effector, Rac, transduced the guidance signal from PVR during border cell migration. Here we demonstrate that PVR is also required for the morphogenetic process, thorax closure, during metamorphosis. The results of genetic and biochemical experiments indicate that PVR activates the JNK pathway. We present evidence showing Crk (an adaptor molecule), Mbc, ELMO (a homolog of Caenorhabditis elegans CED-12 and mammalian ELMO), and Rac to be mediators of JNK activation by PVR. In addition, we suppose that not only Rac but also Cdc42 is activated and involved in JNK activation downstream of PVR.

Cell Cycle Effects by C-FADD Depend on Its C-terminal Phosphorylation Site

Expression of a truncated form of the death receptor adaptor FADD (C-FADD) as a transgene in mice blocks T cell proliferation. Here we provide evidence that the C-terminal phosphorylation site Ser194 in C-FADD affects the cell cycle in nonlymphoid cells as well. High expression of wild type C-FADD but not C-FADD with a S194A point mutation arrested the nontumor cell line MCF10A in G2/M but not the tumor cell line MCF7. BJAB as well as MCF10A cells expressing moderate levels of C-FADD with a S194E mutation mimicking phosphorylated C-FADD were more susceptible to a Taxol-induced G2/M arrest than cells expressing C-FADD S194A suggesting that C-FADD S194E lowers the threshold for G2/M arrest. Our data suggest that C-FADD may affect apoptosis sensitivity of cells by interfering with cell cycle progression and not only by binding to death receptors.

The association of cyclin A and cyclin kinase inhibitor p21 in response to γ-irradiation requires the CDK2 binding region, but not the Cy motif

The cyclin kinase inhibitor p21 associates with and inhibits cyclin–CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1 (18CRRL) or Cy2 (154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10 Gy of γ-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to γ-irradiation requires the CDK binding region, 49–71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin–CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the association of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 aa to allow cyclin A association.