Clathrin-dependent association of CVAK104 with endosomes and the trans-Golgi network

CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the beta2-subunit of the adaptor protein (AP) complex AP2 in vitro. Here, we demonstrate that a C-terminal segment of CVAK104 interacts with the N-terminal domain of clathrin and with the alpha-appendage of AP2. CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells, but it is also present on peripheral vesicular structures that are accessible to endocytosed transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor, and clathrin but not at all with its putative phosphorylation target AP2. RNA interference-mediated clathrin knockdown reduced the membrane association of CVAK104. Recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by guanosine 5'-O-(3-thio)triphosphate, and brefeldin A redistributes CVAK104 in cells. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of green fluorescent protein-CVAK104 with endocytosed transferrin and with red fluorescent protein-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D. Together, our data suggest a function for CVAK104 in clathrin-dependent pathways between the trans-Golgi network and the endosomal system.     

CFBP is a novel tyrosine-phosphorylated protein that might function as a regulator of CIN85/CD2AP

To decipher the global network of the epidermal growth factor (EGF) receptor-mediated signaling pathway, a large scale proteomic analysis of tyrosine-phosphorylated proteins was conducted. Here, we focus on characterizing a novel protein, CFBP (CIN85/CD2AP family binding protein), identified in the study. CFBP was found to be phosphorylated at tyrosine 204 upon EGF stimulation, and the CIN85/CD2AP family was identified as a binding partner. A proline-rich motif of CFBP is recognized by one of the three Src-homology 3 domains of CIN85/CD2AP, and the affinity of the interaction is regulated by the tyrosine phosphorylation of CFBP. They co-localize in actinenriched structures, and overexpression of CFBP induced morphological changes with actin reorganization. Furthermore, CFBP accelerated the EGF receptor's down-regulation by facilitating the recruitment of Cbl to the CD2AP/CIN85 complex. Two spliced variants of CFBP lacking either exon 5 or 8 are also expressed, and the variant lacking exon 5 without the proline-rich motif lacks the ability to bind to the CIN85/CD2AP family. The CFBP protein seems to play a key role in the ligand-mediated internalization and down-regulation of the EGF receptor.     

A novel ubiquitin-like domain in IkappaB kinase beta is required for functional activity of the kinase

Activation of NF-kappaB requires two highly related kinases named IKKalpha and IKKbeta that share identity in the nature and positioning of their structural domains. Despite their similarity, the kinases are functionally divergent, and we therefore sought to identify any structural features specific for IKKalpha or IKKbeta. We performed bioinformatics analysis, and we identified a region resembling a ubiquitin-like domain (UBL) that exists only in IKKbeta and that we named the UBL-like domain (ULD). Deletion of the ULD rendered IKKbeta catalytically inactive and unable to induce NF-kappaB activity, and overexpression of only the ULD dose-dependently inhibited tumor necrosis factor-alpha-induced NF-kappaB activity. The ULD could not be functionally replaced within IKKbeta by ubiquitin or the corresponding region of IKKalpha, whereas deletion of the equivalent section of IKKalpha did not affect its catalytic activity against IkappaBalpha or its activation by NF-kappaB-inducing kinase. We identified five residues conserved among the larger family of UBL-containing proteins and IKKbeta, and alanine scanning revealed that the leucine at position 353 (Leu(353)) is absolutely critical for IKKbeta-induced NF-kappaB activation. Most intriguingly, the L353A mutant was catalytically active but, unlike wild-type IKKbeta, formed a stable complex with the NF-kappaB p65 subunit. Our findings therefore establish the ULD as a critical functional domain specific for IKKbeta that might play a role in dissociating IKKbeta from p65.     

Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation.

We have localized a G protein activator region of the human beta 2-adrenergic receptor to region beta III-2 (from Arg259 to Lys273). The synthetic beta III-2, corresponding to the C-terminal end of the third cytoplasmic loop, activates Gs at nanomolar concentrations and weakly activates Gi. beta III-2 activates adenylyl cyclase at nanomolar concentrations in wild-type S49 lymphoma membranes, but not in membranes of unc mutant S49 cells, in which Gs is uncoupled from beta-adrenergic stimulation. Phosphorylation of beta III-2 by cAMP-dependent protein kinase A, which is involved in the desensitization of the beta-adrenergic receptor from Gs, drastically reduces the effect of beta III-2 on Gs while potentiating its action on Gi, resulting in a total loss of adenylyl cyclase-stimulating activity. These findings indicate that this receptor sequence is a multipotential G protein activator whose G protein specificity is regulated by protein kinase A.     

Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3beta

Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.     

cDNA sequence of the beta 2-subunit of human liver alcohol dehydrogenase

A cDNA library of mRNA from a human liver expressing the beta 2-subunit of alcohol dehydrogenase was constructed in lambda gt11. One clone coding for 352 of a total of 374 amino acid residues of the beta 2-subunit was isolated. The sequence differed from that of the beta 1-subunit at one nucleotide position resulting in an Arg/His exchange at position 47 of the peptide chain, in agreement with data from protein sequence analysis [(1984) FEBS Lett. 173, 360-366].     

WNK2 kinase is a novel regulator of essential neuronal cation-chloride cotransporters

NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl(-)]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl(-)-dependent (86)Rb(+) uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl(-) accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO(2) enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling.     

Ca2+/Calmodulin-dependent protein kinase kinase beta is regulated by multisite phosphorylation

Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a serine/threonine-directed kinase that is activated following increases in intracellular Ca(2+). CaMKKβ activates Ca(2+)/calmodulin-dependent protein kinase I, Ca(2+)/calmodulin-dependent protein kinase IV, and the AMP-dependent protein kinase in a number of physiological pathways, including learning and memory formation, neuronal differentiation, and regulation of energy balance. Here, we report the novel regulation of CaMKKβ activity by multisite phosphorylation. We identify three phosphorylation sites in the N terminus of CaMKKβ, which regulate its Ca(2+)/calmodulin-independent autonomous activity. We then identify the kinases responsible for these phosphorylations as cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). In addition to regulation of autonomous activity, we find that phosphorylation of CaMKKβ regulates its half-life. We find that cellular levels of CaMKKβ correlate with CDK5 activity and are regulated developmentally in neurons. Finally, we demonstrate that appropriate phosphorylation of CaMKKβ is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKKβ-dependent signaling cascades.     

Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine-phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (amino acids 106-206) revealed that it forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as E-cadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features.     

The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival

NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.     

Constitutive GABAA receptor endocytosis is dynamin-mediated and dependent on a dileucine AP2 adaptin-binding motif within the beta 2 subunit of the receptor

Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA(A) receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA(A) receptors composed of either alpha1beta2gamma2Lor alpha1beta2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor beta2 subunit that is critical for endocytosis. Internalization of GABAA receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor beta2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA(A) receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.     

SRPX2 is a novel chondroitin sulfate proteoglycan that is overexpressed in gastrointestinal cancer

SRPX2 (Sushi repeat-containing protein, X-linked 2) has recently emerged as a multifunctional protein that is involved in seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was secreted with a highly posttranslational modification. Chondroitinase ABC treatment completely decreased the molecular mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel chondroitin sulfate proteoglycan (CSPG). Furthermore, a binding assay revealed that hepatocyte growth factor dose-dependently binds to SRPX2 protein, and a ligand-glycosaminoglycans interaction was speculated to be likely in proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans; however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related proteoglycan family.     

Characterization of AP3B2_v2, a novel splice variant of human AP3B2.

A novel splice variant of human AP3B2, named AP3B2_v2, was isolated through the large-scale sequencing analysis of a human fetal brain cDNA library. The AP3B2_v2 cDNA is 1171 bp in length. Sequence analysis revealed AP3B2_v2 missed 22 exons that existed in AP3B2_v1, leading to a different putative protein. The deduced proteins were 145 amino acids (designated as AP3B2_v2) and 1082 amino acids (AP3B2_v1) in length, sharing the C-terminal 145 amino acids. RT-PCR analysis showed that human AP3B2_v2 were expressed in several human adult tissues analyzed. The expression levels of AP3B2_v2 were relatively high in brain and testis. In contrast, low levels of expression were detected in kidney, pancreas, spleen, thymus, prostate, ovary and small intestine.     

MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3

The MCM proteins are essential for the initiation of DNA replication. We have isolated an MCM3-associated protein (MCM3AP) in a two-hybrid screen using MCM3. Here we demonstrate that MCM3AP is an acetyltransferase which acetylates MCM3 and that chromatin-bound MCM3 is acetylated in vivo. The MCM3 acetylase, MCM3AP, is also chromatin-bound. This study also indicates that MCM3AP contains putative acetyl CoA binding motifs conserved within the GCN5-related N-acetyltransferase superfamily. Mutation of those motifs significantly inhibits the MCM3 acetylase activity. Over-expression of MCM3AP inhibits DNA replication, whereas mutation of the acetylase motifs abolishes this effect, suggesting that acetylation plays a role in DNA replication. Taken together, we suggest that MCM3 acetylation is a novel pathway which might regulate DNA replication.     

Binding of AP2 to sorting signals is modulated by AP2 phosphorylation

The two clathrin-associated adaptor complexes AP1 and AP2 are known to participate in the formation of clathrin-coated vesicles at the trans-Golgi network and at the plasma membrane. During this process adaptors are involved in the sequestration of vesicle cargo by binding to the sorting signals within the cytoplasmic domains of the cargo proteins and in the recruitment of the clathrin coat. After budding of the clathrin-coated vesicles, the clathrin and adaptors dissociate from the vesicles. Here we show that in vitro binding of AP2 to sorting signals, which is one of the initial steps in receptor-mediated endocytosis, is modulated by adaptor phosphorylation. AP2 was phosphorylated by incubating purified AP2 in the presence of ATP and dephosphorylated by incubation with alkaline phosphatase. Affinity for tyrosine-, leucine-based and noncanonical sorting motifs was 15-33 times higher for phosphorylated than for dephosphorylated AP2. Also the binding of AP2 to membranes was regulated by adaptor phosphorylation/dephosphorylation and was about 8-fold higher for phosphorylated than for dephosphorylated AP2. Moreover, AP2 isolated from cytosol is higher phosphorylated than membrane-extracted and exhibits a 5-fold higher binding affinity than AP2 extracted from membranes. Taken together these data point to a cycle of phosphorylation/dephosphorylation as a mechanism for regulating the reversible association of AP2 with membranes and sorting signals during the process of receptor-mediated endocytosis.     

The lysine-specific demethylase 1 is a novel substrate of protein kinase CK2

Protein kinase CK2 is a pleiotropic serine/threonine kinase responsible for the generation of a substantial proportion of the human phosphoproteome. CK2 is generally found as a tetramer with two catalytic, α and α′ and two non catalytic β subunits. CK2α C-terminal tail phosphorylation is regulated during the mitotic events and the absence of these phosphosites in α′ suggests an isoform specialization. We used a proteomic approach to identify proteins specifically phosphorylated by a CK2α phosphomimetic mutant, CK2αT344ET360ES362ES370E (CK2α4E), in human neuroblastoma SKNBE cellular extract. One of these proteins is lysine-specific demethylase 1 (LSD1 or KDM1A), an important player of the epigenetic machinery. LSD1 is a FAD-dependent amine oxidase and promotes demethylation of lysine 4 and lysine 9 of mono- and di-methylated histone H3. We found that LSD1 is a new substrate and an interacting partner of protein kinase CK2. Three CK2 phosphosites, (Ser131, Ser137 and Ser166) in the N-terminal region of LSD1 have been identified. This domain is found in all chordates but not in more ancient organisms and it is not essential for LSD1 catalytic event while it could modulate the interaction with CK2 and with other partners in gene repressing and activating complexes. Our data support the view that the phosphorylation of the N-terminal domain by CK2 may represent a mechanism for regulating histone methylation, disclosing a new role for protein kinase CK2 in epigenetics.