Signaling adaptor protein Crk is indispensable for malignant feature of glioblastoma cell line KMG4

Signaling adaptor protein Crk has been shown to be involved in pathogenesis of human cancers including brain tumor where Crk was reported to be overexpressed. In this study, we addressed whether Crk is indispensable for malignant phenotype of brain tumor. In 20 surgical specimens of glioma, mRNA of both CrkI and CrkII was found to be elevated in malignant tumor. To define a precise role of Crk, we have established Crk-knockdown cell lines of glioblastoma KMG4 by siRNA, and early phase of cell adhesion to laminin was found to be suppressed. Wound healing assay revealed the decreased cell motility in Crk knockdown cells, and suppression of both anchorage-dependent and -independent growth were demonstrated in these cells. Furthermore, in vivo tumor forming potential was also markedly suppressed. These results suggest that Crk is required for early attachment to laminin, cell motility, and growth of glioblastoma cell line KMG4.     

The substrate specificity of the catalytic domain of Abl plays an important role in directing phosphorylation of the adaptor protein Crk

c-Abl preferentially phosphorylates peptide substrates that contain proline at the P+3 site (relative to the phosphorylated tyrosine). We previously described a mutant form of the Abl catalytic domain (Y569W) with altered substrate specificity at the P+3 position, as measured using synthetic peptides. In this study, we examine the phosphorylation of Crk, a protein substrate of Abl that is phosphorylated in the sequence Tyr221-Ala-Gln-Pro. In vitro, phosphorylation of Crk by Y569W Abl is greatly reduced relative to wild-type Abl. Overexpression of Y569W mutant Abl in 293T kidney cells produces a similar overall pattern of tyrosine phosphorylation as wild-type Abl, indicating that not all cellular proteins depend on Pro at P+3 for Abl recognition. However, phosphorylation of Crk by Y569W Abl in these cells is markedly reduced relative to wild-type Abl. A truncated form of Abl lacking the C-terminal polyproline region is not able to phosphorylate Crk in these assay conditions. Thus, proper phosphorylation of Crk by Abl depends not only on the interaction of the Crk SH3 domain with the Abl polyproline region, but also on the recognition of amino acids surrounding tyrosine by the Abl catalytic domain.     

Crk and CrkL adaptor proteins: networks for physiological and pathological signaling

The Crk adaptor proteins (Crk and CrkL) constitute an integral part of a network of essential signal transduction pathways in humans and other organisms that act as major convergence points in tyrosine kinase signaling. Crk proteins integrate signals from a wide variety of sources, including growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. Mounting evidence indicates that dysregulation of Crk proteins is associated with human diseases, including cancer and susceptibility to pathogen infections. Recent structural work has identified new and unusual insights into the regulation of Crk proteins, providing a rationale for how Crk can sense diverse signals and produce a myriad of biological responses.     

SRC-like adaptor protein regulates B cell development and function

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.     

The Rad9A checkpoint protein is required for nuclear localization of the claspin adaptor protein

The interaction between the 911 complex, via Rad9A, and Claspin is required for activation of the Chk1-mediated checkpoint response, along with ATR, TopBP1, and the 911 clamp loader complex Rad17/RFC. Despite the importance of the Rad9A-Claspin interaction in the cell cycle, this interaction has yet to be characterized. In this work we show this interaction persists in a variety of different conditions. During the course of this study we also determined the nuclear localization of Rad9A affected the localization of the Claspin protein, leading us to the conclusion that Rad9A is able to affect Claspin cellular localization. This was verified experimentally using a Rad9A-null cell line and reconstitution of WT Rad9A. We also show that in mES cells the Rad9A paralog, Rad9B, is also capable of affecting Claspin localization. Together, these data suggest that Rad9 plays a role in locating Claspin to sites of DNA damage, facilitating its role during the Chk1-mediated checkpoint response. Since disruption of both Rad9A and Claspin has been shown to abolish Chk1 activation, we postulate that Rad9A-mediated Claspin localization is a vital step during checkpoint activation.     

The function of the nuclear envelope in nuclear protein accumulation

The mechanism by which proteins accumulate in the cell nucleus is not yet known. Two alternative mechanisms are discussed here: (a) selective unidirectional entry of karyophilic proteins through the nuclear pores, and (b) free diffusion of all proteins through the nuclear pores and specific binding of nuclear proteins to nondiffusible components of the nucleoplasm. We present experiments designed to distinguish between these alternatives. After mechanical injury of the Xenopus oocyte nuclear envelope, nuclear proteins were detected in the cytoplasm by immunohistochemical methods. In a second approach, nuclei from X. borealis oocytes were isolated under oil, the nuclear envelopes were removed, and the pure nucleoplasm was injected into the vegetal pole of X. laevis oocytes. With immunohistochemical methods, it was found that each of five nuclear proteins rapidly diffuses out of the injected nucleoplasm into the surrounding cytoplasm. The subsequent transport and accumulation in the intact host nucleus could be shown for the nuclear protein N1 with the aid of a species-specific mAb that reacts only with X. borealis N1. Purified and iodinated nucleoplasmin was injected into the cytoplasm of Xenopus oocytes and its uptake into the nucleus was studied by biochemical methods.     

Structure and function of ALG-2, a penta-EF-hand calcium-dependent adaptor protein

ALG-2 (a gene product of PDCD6) is a 22-kD protein containing five serially repetitive EF-hand structures and belongs to the penta-EF-hand (PEF) family, including the subunits of typical calpains. ALG-2 is the most conserved protein among the PEF family members and its homologs are widely found in eukaryotes. X-ray crystal structures of various PEF proteins including ALG-2 have common features: presence of eight α-helices and dimer formation via paired EF5s that are positioned in anti-parallel orientation. ALG-2 forms a homodimer and a heterodimer with its closest paralog peflin. Like calmodulin, a well-known four-EF-hand protein, ALG-2 interacts with various proteins in a Ca²⁺-dependent fashion, but the binding motifs are completely different. With some exceptions, ALG-2-interacting proteins commonly contain Pro-rich regions, and ALG-2 recognizes at least two distinct Pro-containing motifs: PPYP(X)nYP (X, variable; n=4 in ALIX and PLSCR3) and PXPGF (represented by Sec31A). A shorter alternatively spliced isoform, lacking two residues and designated ALG-2^ΔGF122, does not bind ALIX but maintains binding capacity to Sec31A. X-ray crystal structural analyses have revealed that binding of calcium ions induces the configuration of the side chain of R125 so that it opens Pocket 1, which accepts PPYP, but Pocket 1 remains closed in the case of ALG-2^ΔGF122. ALG-2 dimer has two ligand-binding sites, each in a monomer molecule, and appears to function as a Ca²⁺-dependent adaptor protein to either stabilize a preformed complex or to bridge two proteins on scaffolds in systems of the endosomal sorting complex required for transport (ESCRT) and ER-to-Golgi transport.     

Structure of the adaptor protein p14 reveals a profilin-like fold with distinct function

The adaptor protein p14 is associated with the cytoplasmic face of late endosomes that is involved in cell-surface receptor endocytosis and it also directly interacts with MP1, a scaffolding protein that binds the MAP kinase ERK1 and its upstream kinase activator MEK1. The interaction of p14 with MP1 recruits the latter to late endosomes and the endosomal localization of p14/MP1-MEK1-ERK1 scaffolding complex is required for signaling via ERK MAP kinase in an efficient and specific manner upon receptor stimulation. Here, we report the three-dimensional solution structure of the adaptor protein p14. The structure reveals a profilin-like fold with a central five-stranded beta-sheet sandwiched between alpha-helices. Unlike profilin, however, p14 exhibits weak interaction with selective phosphoinositides but no affinity towards proline-rich sequences. Structural comparison between profilin and p14 reveals the molecular basis for the differences in these functions. We further mapped the MP1 binding sites on p14 by NMR, and discuss the implications of these important findings on the possible function of p14.     

Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.     

Negative regulation of Fc epsilonRI-mediated signaling and mast cell function by the adaptor protein LAX

LAX is a transmembrane adaptor protein that is expressed in both T and B cells. Upon stimulation via the antigen receptors, it is tyrosine-phosphorylated and binds Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. Disruption of the Lax gene causes hyperresponsiveness in T and B lymphocytes. Here, we showed that LAX was also expressed in mast cells. Upon engagement of the Fc epsilonRI, LAX was also phosphorylated and interacted with Grb2 and p85. LAX-deficient mast cells were hyperresponsive to stimulation via the Fc epsilonRI, as evidenced by enhanced degranulation, p38 MAPK, Akt, and phosphatidylinositol 3-kinase activation. This hyperresponsiveness was likely a consequence of reduced LAB expression after sensitization of mast cells with anti-dinitrophenyl IgE. In addition, Fc epsilonRI-mediated cytokine production and cell survival were also enhanced. These data suggested that LAX negatively regulates mast cell function.     

Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta

The goal of this study was to determine whether the intracellular distribution of the proapoptotic enzyme glycogen synthase kinase-3 beta (GSK-3 beta) is dynamically regulated by conditions that activate apoptotic signaling cascades. In untreated human neuroblastoma SH-SY5Y cells, GSK-3 beta was predominantly cytosolic, although a low level was also detected in the nucleus. The nuclear level of GSK-3 beta was rapidly increased after exposure of cells to serum-free media, heat shock, or staurosporine. Although each of these conditions caused changes in the serine 9 and/or tyrosine phosphorylation of GSK-3 beta, neither of these modifications was correlated with nuclear accumulation of GSK-3 beta. Heat shock and staurosporine treatments increased nuclear GSK-3 beta prior to activation of caspase-9 and caspase-3, and this nuclear accumulation of GSK-3 beta was unaltered by pretreatment with a general caspase inhibitor. The GSK-3 beta inhibitor lithium did not alter heat shock-induced nuclear accumulation of GSK-3 beta but increased the nuclear level of cyclin D1, indicating that cyclin D1 is a substrate of nuclear GSK-3 beta. Thus, the intracellular distribution of GSK-3 beta is dynamically regulated by signaling cascades, and apoptotic stimuli cause increased nuclear levels of GSK-3 beta, which facilitates interactions with nuclear substrates.     

Proapoptotic function of protein kinase CK2alpha" is mediated by a JNK signaling cascade

Protein kinase CK2 (formerly casein kinase II) is a tetrameric enzyme constitutively expressed in all eurakyotic tissues that plays a significant role in the regulation of cell proliferation, malignant transformation, and apoptosis. The catalytic alpha-subunit of the enzyme is known to exist in three isoforms CK2alpha, CK2alpha' and CK2alpha". CK2alpha" is highly expressed in liver compared with other tissues and is required for the normal trafficking of several hepatocellular membrane proteins. Initial studies of dengue virus infection indicated that the CK2alpha"-deficient membrane trafficking mutant cell line (Trf1) was resistant to virus-induced cell death compared with the parental human hepatoma (HuH)-7 hepatoma line. Expression of recombinant CK2alpha" in Trf1 was capable of reverting this resistant phenotype. This study was extended to TNF-alpha in addition to other stimuli of cell death in an attempt to uncover common death pathways that might be modulated by CK2alpha". Evaluation of different pathways involved in death signaling suggest that the regulation of a critical proapoptotic step in HuH-7 cells by CK2alpha" is mediated by a JNK signaling cascade.     

Fibroblast growth factor receptor-1-mediated endothelial cell proliferation is dependent on the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) expressed on endothelial cells leads to cellular migration and proliferation. We have examined the role of the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk in these processes. Transient tyrosine phosphorylation of Crk in fibroblast growth factor-2-stimulated endothelial cells was dependent on the juxtamembrane tyrosine residue 463 in FGFR-1, and a Crk SH2 domain precipitated FGFR-1 via phosphorylated Tyr-463, indicating direct complex formation between Crk and FGFR-1. Furthermore, Crk SH2 and SH3 domains formed ligand-independent complexes with Shc, C3G, and the Crk-associated substrate (Cas). Tyrosine phosphorylation of C3G and Cas increased as a consequence of growth factor treatment. We examined the role of Crk in FGFR-1-mediated cellular responses by use of cells expressing chimeric platelet-derived growth factor receptor-alpha/FGFR-1 (alphaR/FR) wild type and mutant Y463F receptors. The kinase activity of alphaR/FR Y463F was intact, but both Crk and the adaptor FRS-2 were no longer tyrosine-phosphorylated in the mutant cells. Both wild type and mutant receptor cells migrated efficiently, whereas cells expressing the mutant alphaR/FR Y463F failed to proliferate and Erk2 and Jun kinase activities were suppressed in these cells. In wild type alphaR/FR cells transiently expressing an SH2 domain mutant of Crk, Erk and Jun kinase activities as well as DNA synthesis were attenuated. Our data indicate that Crk participates in signaling complexes downstream of FGFR-1, which propagate mitogenic signals.     

Proapoptotic function of the MET tyrosine kinase receptor through caspase cleavage

The MET tyrosine kinase, the receptor of hepatocyte growth factor-scatter factor (HGF/SF), is known to be essential for normal development and cell survival. We report that stress stimuli induce the caspase-mediated cleavage of MET in physiological cellular targets, such as epithelial cells, embryonic hepatocytes, and cortical neurons. Cleavage occurs at aspartic residue 1000 within the SVD site of the juxtamembrane region, independently of the crucial docking tyrosine residues Y1001 or Y1347 and Y1354. This cleavage generates an intracellular 40-kDa MET fragment containing the kinase domain. The p40 MET fragment itself causes apoptosis of MDCK epithelial cells and embryonic cortical neurons, whereas its kinase-dead version is impaired in proapoptotic activity. Finally, HGF/SF treatment does not favor MET cleavage and apoptosis, confirming the known survival role of ligand-activated MET. Our results show that stress stimuli convert the MET survival receptor into a proapoptotic factor.     

The fifth adaptor protein complex

Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.     

Analysis of the adaptor function of the LIM domain-containing protein FHL2 using an affinity chromatography approach

Containing four LIM domains and an N-terminal half LIM domain, FHL2 has been predicted to have an adaptor function in the formation of higher order molecular complexes in the nucleus and the cytoplasm of cells. We expressed recombinant FHL2 in insect cells using the baculovirus system and used it to isolate direct or indirect interaction partners from the cytosolic fraction of fibroblasts by affinity chromatography. These were identified by their peptide mass fingerprints using MALDI-TOF mass spectrometry. Cytoskeleton-associated proteins present among the bound proteins were shown to co-localise with FHL2 in cell lamellipodia by indirect immunofluorescence staining.