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PubMed2003

Affinity purification of PSD-95-containing postsynaptic complexes.

A widely used method for the preparation of postsynaptic density (PSD) fractions consists of treatment of synaptosomal membranes with Triton X-100 and further purification by density gradient centrifugation. In the present study, the purity of this preparation was assessed by electron microscopic analysis. Thin-section and rotary shadow immuno-electron microscopy of the Triton X-100-derived PSD fraction shows many PSD-95-positive structures that resemble in situ PSDs in shape and size. However, the fraction also includes contaminants such as CaMKII clusters, spectrin filaments and neurofilaments. We used magnetic beads coated with an antibody against PSD-95 to further purify PSD-95-containing complexes from the Triton-derived PSD fraction. Biochemical analysis of the affinity-purified material shows a substantial reduction in the astrocytic marker glial fibrillary acidic protein and electron microscopic analysis shows mostly individual PSDs attached to magnetic beads. This preparation was used to assess the association of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptors with the PSD-95-containing complex. AMPA receptors are demonstrated by immunoblotting to be present in the complex, although they do not co-purify exclusively with PSD-95, suggesting the existence of two pools of receptors, one associated with the PSD-95 scaffold and the other not. Of the AMPA receptor-anchoring proteins tested, SAP-97 is present in the affinity-purified preparation whereas GRIP is found only in trace amounts. These results imply that a subpopulation of AMPA receptors is anchored to the PSD-95-containing scaffold through interaction of GluR1 with SAP-97.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Increased vulnerability to kainic acid-induced epileptic seizures in mice underexpressing the scaffold protein Islet-Brain 1/JIP-1.

Islet-Brain 1, also known as JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein mainly involved in the regulation of the pro-apoptotic signalling cascade mediated by c-Jun-N-terminal kinase (JNK). IB1/JIP-1 organizes JNK and upstream kinases in a complex that facilitates JNK activation. However, overexpression of IB1/JIP-1 in neurons in vitro has been reported to result in inhibition of JNK activation and protection against cellular stress and apoptosis. The occurrence and the functional significance of stress-induced modulations of IB1/JIP-1 levels in vivo are not known. We investigated the regulation of IB1/JIP-1 in mouse hippocampus after systemic administration of kainic acid (KA), in wild-type mice as well as in mice hemizygous for the gene MAPK8IP1, encoding for IB1/JIP-1. We show here that IB1/JIP-1 is upregulated transiently in the hippocampus of normal mice, reaching a peak 8 h after seizure induction. Heterozygous mutant mice underexpressing IB1/JIP-1 showed a higher vulnerability to the epileptogenic properties of KA, whereas hippocampal IB1/JIP-1 levels remained unchanged after seizure induction. Subsequently, an increasing activation of JNK in the 8 h following seizure induction was observed in IB1/JIP-1 haploinsufficient mice, which also underwent more severe excitotoxic lesions in hippocampal CA3, as assessed histologically 3 days after KA administration. Taken together, these data indicate that IB1/JIP-1 in hippocampus participates in the regulation of the neuronal response to excitotoxic stress in a level-dependent fashion.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Caspase-8 regulation by direct interaction with TRAF6 in T cell receptor-induced NF-kappaB activation.

Triggering of lymphocyte antigen receptors is the critical first step in the adaptive immune response against pathogens. T cell receptor (TCR) ligation assembles a large membrane signalosome, culminating in NF-kappaB activation [1,2]. Recently, caspase-8 was found to play a surprisingly prominent role in lymphocyte activation in addition to its well-known role in apoptosis [3]. Caspase-8 is activated after TCR stimulation and nucleates a complex with B cell lymphoma 10 (BCL10), paracaspase MALT1, and the inhibitors of kappaB kinase (IKK) complex [4]. We now report that the ubiquitin ligase TRAF6 binds to active caspase-8 upon TCR stimulation and facilitates its movement into lipid rafts. We identified in silico two putative TRAF6 binding motifs in the caspase-8 sequence and found that mutation of critical residues within these sites abolished TRAF6 binding and diminished TCR-induced NF-kappaB activation. Moreover, RNAi-mediated silencing of TRAF6 abrogated caspase-8 recruitment to the lipid rafts. Protein kinase Ctheta (PKCtheta), CARMA1, and BCL10 are also required for TCR-induced caspase-8 relocation, but only PKCtheta and BCL10 control caspase-8 activation. Our results suggest that PKCtheta independently controls CARMA1 phosphorylation and BCL10-dependent caspase-8 activation and unveil an essential role for TRAF6 as a critical adaptor linking these two convergent signaling events.

Amino Acid Motifs↗
PubMed2006

Activation of the nonreceptor protein tyrosine kinase Ack by multiple extracellular stimuli.

Ack/Ack1 is a nonreceptor protein tyrosine kinase that comprises a tyrosine kinase core, an SH3 domain, a Cdc42-binding region, a Ralt homology region, and a proline-rich region. Here we describe a detailed characterization of the Ack protein as well as the chromosomal localization of human Ack (chromosome 3q29) and the primary structure of murine Ack. We demonstrate that Ack is ubiquitously expressed, with highest expression seen in thymus, spleen, and brain. Activation of integrins by cell adhesion on fibronectin leads to strong tyrosine phosphorylation and activation of Ack. Upon cell stimulation with EGF or PDGF, Ack is tyrosine-phosphorylated and recruited to activated EGF or PDGF receptors, respectively. A pool of endogenous Ack molecules is constitutively tyrosine-phosphorylated, even in starved cells. Moreover, tyrosine-phosphorylated Ack forms a stable complex with the adapter protein Nck via its SH2 domain. Finally, we have characterized a membrane-targeting sterile alpha motif-like domain in the amino terminus of Ack. Using several Ack mutants, we show that the amino-terminal and CRIB domains are necessary for Ack autophosphorylation, whereas the SH3 domain appears to have an autoinhibitory role. These experiments suggest a functional role for Ack as an early transducer of multiple extracellular stimuli.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Structural localization of disease-associated sequence variations in the NACHT and LRR domains of PYPAF1 and NOD2.

Several autoinflammatory diseases with distinct clinical manifestations have been associated with sequence variations in the gene products PYPAF1/CIAS1 and NOD2/CARD15. Both proteins belong to the PYD/CARD-containing family of apoptosis regulators and activators of pro-inflammatory caspases. To gain insight into the dysfunctional role of sequence alterations, we assembled a structure-based multiple sequence alignment of family members and related proteins. This allowed us to analyze the putative effect of the alterations on the function of nucleotide-binding (NACHT) and leucine-rich repeat (LRR) domains shared by the family members. In support of this analysis, we carefully selected template structures for the NACHT and LRR domains and mapped the genetic variations onto 3D domain models. Additionally, we propose a model of the NACHT and LRR domain complex. Our study revealed that many of the disease-associated sequence variants are located close to highly conserved sequence regions of functional relevance and are spatially adjacent in the predicted 3D structure. The implications on the domain functions such as NTP-hydrolysis or oligomerization are discussed.

Amino Acid Sequence↗
PubMed2005

Domain interactions within the Ski2/3/8 complex and between the Ski complex and Ski7p.

The Ski complex (composed of Ski3p, Ski8p, and the DEVH ATPase Ski2p) is a central component of the 3'-5' cytoplasmic mRNA degradation pathway in yeast. Although the proteins of the complex interact with each other as well as with Ski7p to mediate degradation by exosome, a 3'-exonuclease complex, the nature of these interactions is not well understood. Here we explore interactions within the Ski complex and between the Ski complex and Ski7p using a directed two-hybrid approach combined with coimmunoprecipitation experiments. We also test the functional significance of these interactions in vivo. Our results suggest that within the Ski complex, Ski3p serves as a scaffold protein with its C terminus interacting with Ski8p, and the sub-C terminus interacting with Ski2p, while no direct interaction between Ski2p and Ski8p was found. Ski7p interacts with the Ski complex via its interaction with Ski8p and Ski3p. In addition, inactivating the Ski complex by mutating conserved residues in the DEVH helicase motif of Ski2 did not abrogate its interaction with Ski7p, indicating that Ski2p function is not necessary for this interaction.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Intersectin regulates epidermal growth factor receptor endocytosis, ubiquitylation, and signaling.

Receptor tyrosine kinases (RTKs) are critical for normal cell growth, differentiation, and development, but they contribute to various pathological conditions when disrupted. Activation of RTKs stimulates a plethora of pathways, including the ubiquitylation and endocytosis of the receptor itself. Although endocytosis terminates RTK signaling, it has emerged as a requisite step in RTK activation of signaling pathways. We have discovered that the endocytic scaffolding protein intersectin (ITSN) cooperated with epidermal growth factor receptor (EGFR) in the regulation of cell growth and signaling. However, a biochemical link between ITSN and EGFR was not defined. In this study, we demonstrate that ITSN is a scaffold for the E3 ubiquitin ligase Cbl. ITSN forms a complex with Cbl in vivo mediated by the Src homology (SH) 3 domains binding to the Pro-rich COOH terminus of Cbl. This interaction stimulates the ubiquitylation and degradation of the activated EGFR. Furthermore, silencing ITSN by RNA interference attenuated EGFR internalization as well as activation of the extracellular signal-regulated kinasemitogen-activated protein kinase pathway, thereby demonstrating the importance of ITSN in EGFR function. Given the cooperativity between ITSN and additional RTKs, these results point to an important evolutionarily conserved, regulatory role for ITSN in RTK function that is necessary for both signaling from receptors as well as the ultimate termination of receptor signaling.

Adaptor Proteins, Vesicular Transport↗
PubMed2002

Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites.

In cells, molecular motors operate in polarized sorting of molecules, although the steering mechanisms of motors remain elusive. In neurons, the kinesin motor conducts vesicular transport such as the transport of synaptic vesicle components to axons and of neurotransmitter receptors to dendrites, indicating that vesicles may have to drive the motor for the direction to be correct. Here we show that an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor subunit--GluR2-interacting protein (GRIP1)--can directly interact and steer kinesin heavy chains to dendrites as a motor for AMPA receptors. As would be expected if this complex is functional, both gene targeting and dominant negative experiments of heavy chains of mouse kinesin showed abnormal localization of GRIP1. Moreover, expression of the kinesin-binding domain of GRIP1 resulted in accumulation of the endogenous kinesin predominantly in the somatodendritic area. This pattern was different from that generated by the overexpression of the kinesin-binding scaffold protein JSAP1 (JNK/SAPK-associated protein-1, also known as Mapk8ip3), which occurred predominantly in the somatoaxon area. These results indicate that directly binding proteins can determine the traffic direction of a motor protein.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Role of protein kinase C in signal attenuation following T cell receptor engagement.

T lymphocyte activation through stimulation of the T cell receptor complex and co-stimulatory receptors is associated with acute tyrosine phosphorylation of intracellular proteins, which in turn mediate downstream signaling events that regulate interleukin-2 expression and cell proliferation. The extent of protein tyrosine phosphorylation is rapidly attenuated after only 1-2 min of stimulation as a means of tightly controlling the initial signaling response. Here we show that this attenuation of tyrosine phosphorylation of Shc, CrkL, and the proto-oncogene Cbl is mimicked by treatment of mouse T lymphocytes or cultured Jurkat cells with phorbol 12-myristate 13-acetate. This effect is blocked by the specific protein kinase C inhibitor GF109203X, but not by PD98059, an inhibitor of MEK1/2 kinase. Activation of protein kinase C by phorbol ester also causes rapid (t(1)/(2) = 2 min) dissociation of both CrkL and p85/phosphoinositide 3-kinase from Cbl concomitant with Cbl tyrosine dephosphorylation. More important, GF109203X treatment of Jurkat cells prior to T cell receptor stimulation by anti-CD3/CD4 antibodies results in an enhanced (2-fold) peak of Cbl phosphorylation compared with that observed in control cells. Furthermore, the rate of attenuation of both Cbl tyrosine phosphorylation and its association with CrkL following stimulation with anti-CD3/CD4 antibodies is much slower in Jurkat cells treated with GF109203X. Taken together, these data provide strong evidence that one or more isoforms of phorbol ester-responsive protein kinase C play a key role in a feedback mechanism that attenuates tyrosine phosphorylation of proteins and reverses formation of signaling complexes in response to T cell receptor activation.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Structure of the N-terminal SH3 domain of GRB2 complexed with a peptide from the guanine nucleotide releasing factor Sos.

Src-homology 3 (SH3) domains mediate signal transduction by binding to proline-rich motifs in target proteins. We have determined the high-resolution NMR structure of the complex between the amino-terminal SH3 domain of GRB2 and a ten amino acid peptide derived from the guanine nucleotide releasing factor Sos. The NMR data show that the peptide adopts the conformation of a left-handed polyproline type II helix and interacts with three major sites on the SH3 domain. The orientation of the bound peptide is opposite to that of proline-rich peptides bound to the SH3 domains of Abl, Fyn and p85.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Platelet endothelial aggregation receptor 1 (PEAR1), a novel epidermal growth factor repeat-containing transmembrane receptor, participates in platelet contact-induced activation.

The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an alpha(IIb)beta(3) antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to alpha(IIb)beta(3)-mediated platelet-platelet contacts.

Amino Acid Sequence↗
PubMed2006

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.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Exit of major histocompatibility complex class II-invariant chain p35 complexes from the endoplasmic reticulum is modulated by phosphorylation.

The Iip35 isoform of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains an endoplasmic reticulum (ER) targeting motif, but in B cell lines the ER retention is ineffective and a fraction of Iip35 is transported through the Golgi complex associated with class II molecules. We found Iip35 (but not Iip33, the major form of Ii) to be phosphorylated in B cell lines, as well as in transfected HeLa cells. The phosphorylation of Iip35 was found to be necessary for the exit of Iip35-class II complexes out of the ER. This requirement suggests that phosphorylation may change the interaction with factors responsible for ER retention/retrieval, and we did find that phosphorylated Iip35 associates with 14-3-3 proteins, a family of adaptor proteins that are involved in coordinating signal transduction pathways. This finding raises the intriguing possibility that the exit of Ii-class II complexes from the ER is regulated by intracellular signaling events.

14-3-3 Proteins↗
PubMed1994

C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins.

CRK protein, together with GRB2/ASH and Nck proteins, belongs to the adaptor-type Src homology (SH)2-containing molecules, which transduce signals from tyrosine kinases. Here another guanine nucleotide-releasing protein (GNRP), C3G, has been identified as a CRK SH3-binding protein. The nucleotide sequence of a 4.1-kb C3G cDNA contains a 3.2-kb open reading frame encoding a 121-kDa protein, and antibodies against C3G have been shown to detect a protein of 130-140 kDa. The carboxyl terminus of C3G has a peptide sequence homologous to GNRPs for Ras, and the expression of this carboxyl terminus region suppresses the loss of CDC25 function in the yeast Saccharomyces cerevisiae. The C3G protein expressed in Escherichia coli binds to CRK and GRB2/ASH proteins. Mutational analysis of C3G assigns the SH3 binding region to a 50-amino acid region containing a proline-rich sequence. The mRNAs of both the C3G and CRK proteins are expressed ubiquitously in human adult and fetal tissues. The results of these studies suggest that the complex of CRK and C3G, or GRB2/ASH and C3G, may transduce the signals from tyrosine kinases to Ras in a number of different tissues.

Adaptor Proteins, Signal Transducing↗
PubMed2005

A pathway for tumor necrosis factor-alpha-induced Bcl10 nuclear translocation. Bcl10 is up-regulated by NF-kappaB and phosphorylated by Akt1 and then complexes with Bcl3 to enter the nucleus.

Bcl10 overexpression and nuclear translocation were originally identified in mucosa-associated lymphoid tissue lymphoma with t(1;14)(p32;q32) chromosome translocation. DNA amplification of Bcl10 was also found in other solid tumors. We have recently shown that nuclear translocation of Bcl10 is a specific molecular determinant of Helicobacter pylori-independent mucosa-associated lymphoid tissue lymphoma (Kuo, S.-H., Chen, L. T., Yeh, K.-H., Wu, M. S., Hsu, H. C., Yeh, P. Y., Mao, T. L., Chen, C. L., Doong, S. L., Lin, J. T., and Cheng, A.-L. (2004) J. Clin. Oncol. 22, 3491-3497). However, the molecular mechanism of Bcl10 nuclear translocation remains unknown. In this study, we observed that tumor necrosis factor-alpha (TNFalpha) up-regulates the expression of Bcl10 and induces a fraction of Bcl10 nuclear translocation in human breast carcinoma MCF7 cells. Chromatin immunoprecipitation assays and electrophoretic mobility shift assays indicated that an NF-kappaB-binding site resides in the Bcl10 5 '-untranslated region. This study also demonstrates that Akt1, activated by TNFalpha, phosphorylates Bcl10 at Ser218 and Ser231 and that phosphorylated Bcl10 subsequently complexes with Bcl3 to enter the nucleus. Either inhibition of Akt1 or depletion of Bcl3 blocks Bcl10 nuclear translocation. In summary, these findings characterize a molecular linkage that directs Bcl10 nuclear translocation in response to TNFalpha treatment.

Active Transport, Cell Nucleus↗
PubMed2004

STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death: multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer.

Using sequence profile analysis and sequence-based structure predictions, we define a previously unrecognized, widespread class of P-loop NTPases. The signal transduction ATPases with numerous domains (STAND) class includes the AP-ATPases (animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators) and NACHT NTPases (e.g. NAIP, TLP1, Het-E-1) that have been studied extensively in the context of apoptosis, pathogen response in animals and plants, and transcriptional regulation in bacteria. We show that, in addition to these well-characterized protein families, the STAND class includes several other groups of (predicted) NTPase domains from diverse signaling and transcription regulatory proteins from bacteria and eukaryotes, and three Archaea-specific families. We identified the STAND domain in several biologically well-characterized proteins that have not been suspected to have NTPase activity, including soluble adenylyl cyclases, nephrocystin 3 (implicated in polycystic kidney disease), and Rolling pebble (a regulator of muscle development); these findings are expected to facilitate elucidation of the functions of these proteins. The STAND class belongs to the additional strand, catalytic E division of P-loop NTPases together with the AAA+ ATPases, RecA/helicase-related ATPases, ABC-ATPases, and VirD4/PilT-like ATPases. The STAND proteins are distinguished from other P-loop NTPases by the presence of unique sequence motifs associated with the N-terminal helix and the core strand-4, as well as a C-terminal helical bundle that is fused to the NTPase domain. This helical module contains a signature GxP motif in the loop between the two distal helices. With the exception of the archaeal families, almost all STAND NTPases are multidomain proteins containing three or more domains. In addition to the NTPase domain, these proteins typically contain DNA-binding or protein-binding domains, superstructure-forming repeats, such as WD40 and TPR, and enzymatic domains involved in signal transduction, including adenylate cyclases and kinases. By analogy to the AAA+ ATPases, it can be predicted that STAND NTPases use the C-terminal helical bundle as a "lever" to transmit the conformational changes brought about by NTP hydrolysis to effector domains. STAND NTPases represent a novel paradigm in signal transduction, whereby adaptor, regulatory switch, scaffolding, and, in some cases, signal-generating moieties are combined into a single polypeptide. The STAND class consists of 14 distinct families, and the evolutionary history of most of these families is riddled with dramatic instances of lineage-specific expansion and apparent horizontal gene transfer. The STAND NTPases are most abundant in developmentally and organizationally complex prokaryotes and eukaryotes. Transfer of genes for STAND NTPases from bacteria to eukaryotes on several occasions might have played a significant role in the evolution of eukaryotic signaling systems.

Adenosine Triphosphatases↗
PubMed2001

Phosphorylation-dependent interactions between ADAM15 cytoplasmic domain and Src family protein-tyrosine kinases.

The adamalysins (ADAMs) are transmembrane glycoproteins involved in cell adhesion and proteolytic ectodomain processing of cytokines and adhesion molecules. Many ADAM cytoplasmic domains are proline-rich and have potential phosphorylation sites. We show here that the cytoplasmic domain of ADAM15, metargidin, can interact specifically with Src family protein-tyrosine kinases (PTKs) and the adaptor protein Grb2 in hematopoietic cells (Jurkat, THP-1, U937, and K562 cell lines). Src homology 3 domains from several Src family PTKs including Lck, Fyn, Abl, and Src associate with ADAM15 in vitro. Dephosphorylation of cell extracts resulted in decreased association of ADAM15 with Src family PTK SH3 domains, indicating that phosphorylation influences ADAM15 interactions with its binding partners. This was confirmed in vitro for Hck, Lck, and Grb2, which showed enhanced association with tyrosine-phosphorylated glutathione S-transferase-ADAM15 cytoplasmic domain compared with unphosphorylated protein. In contrast, binding of MAD2 to ADAM15 was slightly reduced by phosphorylation of the ADAM. Immunoprecipitation of ADAM15 from Jurkat cells confirmed the association with Lck in vivo, and upon PMA stimulation, the phosphorylation level of ADAM15 was increased. Cotransfection of ADAM15 and Hck showed Hck-dependent phosphorylation of ADAM15 in vivo. Hck, and to a lesser extent Lck, phosphorylated the ADAM15 cytoplasmic domain in vitro in immune complex kinase assays. Binding of ADAM15 cytoplasmic domain to Hck and Lck was also shown by Far Western analysis. In contrast to Hck, Lck activity was not required for binding to ADAM15, as shown by treatment of cells with PP1. Deletion and point mutation analysis of the ADAM15 cytoplasmic domain confirmed the importance of the proline-rich motifs for Grb2 and Lck binding and indicated the regulatory nature of Tyr(715) and Tyr(735). These data demonstrate selective, phosphorylation-dependent interactions of ADAM15 with Src family PTKs and Grb2, which highlight the potential for integration of ADAM functions and cellular signaling.

ADAM Proteins↗
PubMed2003

CD2-associated protein and glomerular disease.

CONTEXT: Proteinuria is a major cause of progression in renal disease. The glomerular ultrafiltration barrier, containing highly differentiated podocytes, normally restricts protein access to the urine. Patients with urinary protein in the nephrotic range (>3.5 g daily) often have effaced podocyte foot-processes. Slit diaphragms span the gaps between foot processes as a barrier to macromolecules. Nephrin and podocin are slit-diaphragm proteins identified in families with congenital nephrotic syndromes. CD2-associated protein (CD2AP) is an adapter protein originally identified as a novel ligand interacting with the T-cell-adhesion protein CD2. CD2AP knockout (-/-) mice develop a congenital nephrotic syndrome with podocyte foot-process effacements and die at 6 weeks of age from renal failure. CD2AP localises to the slit diaphragm and links nephrin and podocin to phosphoinositide 3-OH kinase; this complex has cell-signalling properties. STARTING POINT: The CD2AP +/- heterozygous mice developed by Jeong Kim and colleagues (Science 2003; 300: 1298-300) are haploinsufficient and develop glomerular changes at 9 months of age with a histological pattern similar to that in human focal segmental glomerulosclerosis. These researchers found that 2 of 30 African-American patients with idiopathic focal segmental glomerulosclerosis had a CD2AP mutation that ablated expression of one allele. WHERE NEXT? Further studies should address the normal distribution of the CD2AP heterozygous mutation in different ethnic populations, because the association with human idiopathic focal segmental glomerulosclerosis could be accidental. Decreased expression of CD2AP in podocytes of individuals with the CD2AP heterozygous mutation would help to understand how the haploinsufficiency translates into increased susceptibility to renal disease. Transfection of podocytes with mutated CD2AP or study of cultured podocytes from CD2AP +/- mice would provide further insight into whether the nephrin-podocin-CD2AP signal-transduction pathway is altered and leads to increased apoptosis of podocytes. Assuming that a decrease in CD2AP attenuates clearance of glomerular immune complexes, patients with other types of idiopathic glomerulonephritis should also have a CD2AP mutation. However, first studies looking at the most common form of glomerulonephritis, IgA nephropathy, have failed to show decreased renal CD2AP expression.

Adaptor Proteins, Signal Transducing↗