CUB domain-containing protein 1 is a novel regulator of anoikis resistance in lung adenocarcinoma

Malignant tumor cells frequently achieve resistance to anoikis, a form of apoptosis induced by detachment from the basement membrane, which results in the anchorage-independent growth of these cells. Although the involvement of Src family kinases (SFKs) in this alteration has been reported, little is known about the signaling pathways involved in the regulation of anoikis under the control of SFKs. In this study, we identified a membrane protein, CUB-domain-containing protein 1 (CDCP1), as an SFK-binding phosphoprotein associated with the anchorage independence of human lung adenocarcinoma. Using RNA interference suppression and overexpression of CDCP1 mutants in lung cancer cells, we found that tyrosine-phosphorylated CDCP1 is required to overcome anoikis in lung cancer cells. An apoptosis-related molecule, protein kinase Cdelta, was found to be phosphorylated by the CDCP1-SFK complex and was essential for anoikis resistance downstream of CDCP1. Loss of CDCP1 also inhibited the metastatic potential of the A549 cells in vivo. Our findings indicate that CDCP1 is a novel target for treating cancer-specific disorders, such as metastasis, by regulating anoikis in lung adenocarcinoma.     

Identification of the yeast R-SNARE Nyv1p as a novel longin domain-containing protein

Using nuclear magnetic resonance spectroscopy, we establish that the N-terminal domain of the yeast vacuolar R-SNARE Nyv1p adopts a longin-like fold similar to those of Sec22b and Ykt6p. Nyv1p is sorted to the limiting membrane of the vacuole via the adaptor protein (AP)3 adaptin pathway, and we show that its longin domain is sufficient to direct transport to this location. In contrast, we found that the longin domains of Sec22p and Ykt6p were not sufficient to direct their localization. A YXX phi-like adaptin-dependent sorting signal (Y31GTI34) unique to the longin domain of Nyv1p mediates interactions with the AP3 complex in vivo and in vitro. We show that amino acid substitutions to Y31GTI34 (Y31Q;I34Q) resulted in mislocalization of Nyv1p as well as reduced binding of the mutant protein to the AP3 complex. Although the sorting of Nyv1p to the limiting membrane of the vacuole is dependent upon the Y31GTI34 motif, and Y31 in particular, our findings with structure-based amino acid substitutions in the mu chain (Apm3p) of yeast AP3 suggest a mechanistically distinct role for this subunit in the recognition of YXX phi-like sorting signals.     

Mnb/Dyrk1A phosphorylation regulates the interaction of dynamin 1 with SH3 domain-containing proteins

Mnb/Dyrk1A is a proline-directed serine/threonine kinase implicated in Down's syndrome. Mnb/Dyrk1A was shown to phosphorylate dynamin 1 and alter its interactions with several SH3 domain-containing endocytic accessory proteins. To determine the mechanism of regulation, we mapped the Mnb/Dyrk1A phosphorylation sites in dynamin 1. Using a combination of deletion mutants and synthetic peptides, three potential Mnb/Dyrk1A phosphorylation sites (S778, S795, and S857) were first identified. Phosphorylation at S795 and S857 was confirmed in full-length dynamin 1, and S857 was subsequently determined to be the major Mnb/Dyrk1A phosphorylation site in vitro. Phosphorylation at S857 was demonstrated to be the basis for altering the binding of dynamin 1 to amphiphysin 1 and Grb 2 by site-directed mutants mimicking phosphorylation. Furthermore, S857 of dynamin 1 is phosphorylated by the endogenous kinase in brain extracts and in PC12 cells. In PC12 cells, the state of S857 phosphorylation is dependent on membrane potentials. These results suggest that S857 phosphorylation is a physiological event, which regulates the binding of dynamin 1 to SH3 domain-containing proteins. Since S857 is unique to dynamin 1xa isoforms, Mnb/Dyrk1A regulation of dynamin 1 is expected to be specific to these spliced variants.     

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.     

Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.     

CD45 regulates tyrosine phosphorylation of CD22 and its association with the protein tyrosine phosphatase SHP-1

Cross-linking of CD45 induced capping and physical sequestration from CD22 leading to an increase in tyrosine phosphorylation of CD22 and SHP-1 recruitment. Additionally, CD22 isolated from a CD45-deficient B cell line exhibited increased basal/inducible tyrosine phosphorylation and enhanced recruitment of SHP-1 compared with CD22 isolated from CD45-positive parental cells. Subsequent experiments were performed to determine whether enhanced SHP-1 recruitment to CD22 is responsible for attenuation of receptor-mediated Ca2+ responses in CD45-deficient cells. Catalytically inactive SHP-1 expressed in CD45-deficient cells interacted with CD22 and decreased phosphatase activity in CD22 immunoprecipitates to levels that were comparable to those in CD45-positive cells. Expression of catalytically inactive SHP-1 restored intracellular mobilization of Ca2+ in response to MHC class II cross-linking, but did not affect B cell Ag receptor- or class II-mediated Ca2+ influx from the extracellular space. These results indicate that CD45 regulates tyrosine phosphorylation of CD22 and binding of SHP-1. The data further indicate that enhanced recruitment and activation of SHP-1 in CD45-deficient cells affect intracellular mobilization of Ca2+, but are not responsible for abrogation of receptor-mediated Ca2+ influx from the extracellular space.     

Interaction of Axl receptor tyrosine kinase with C1-TEN,a novel C1 domain-containing protein with homology to tensin

Axl receptor tyrosine kinase is implicated in several malignancies and is the receptor for the vitamin K-dependent growth factor Gas6. From a yeast two-hybrid screen of protein-protein interactions with the Axl cytoplasmic domain, we detected a previously uncharacterised SH2 domain-containing protein. We cloned two novel splice variants of this protein that give rise to 1409- and 1419-amino acid proteins, differing only in their N-terminal residues and yielding a 150-kDa protein product by in vitro translation. The Axl-interacting C-terminus contains a tandem SH2 and PTB domain combination homologous to the focal adhesion protein tensin. We detected interaction of Axl with both domains in mammalian cells by co-immunoprecipitation and two-hybrid analyses. In addition, the protein possesses an N-terminal putative phorbol ester-binding C1 domain as well as a central tyrosine phosphatase motif. Thus, we have named the protein C1 domain-containing phosphatase and TENsin homologue (C1-TEN). Northern blot analysis of C1-TEN in human tissues revealed highest expression in heart, kidney, and liver. In summary, we have identified a novel multi-domain intracellular protein that interacts with Axl and which may furthermore be involved in other signal transduction pathways.     

Meiosis-dependent tyrosine phosphorylation of a yeast protein related to the mouse p51ferT

The FER locus of the mouse encodes two mRNA species: one is constitutively transcribed, giving rise to a 94 kDa tyrosine kinase (p94^ferT); the second is a meiosis-specific RNA that gives rise to a 51 kDa tyrosine kinase (p51^ferT). The p51^ferT RNA and protein accumulate in primary spermatocytes that are in prophase of the first meiotic division. By using polyclonal antibodies directed against synthetic peptides derived from the unique amino-terminus of the mouse p51^ferT, a 51 kDa phosphotyrosyl protein — p51^y — was identified in Saccharomyces cerevisiae. The p51^y protein is constitutively expressed in yeast, but in meiotic cells, concomitantly with commitment to meiotic recombination, its level of phosphorylation on tyrosine residues is increased. A different pattern of phosphorylation is observed on serine residues: at early meiotic times the level is decreased, while in later meiotic time the level increases, reaching the vegetative level. When p51^ferT is ectopically expressed in yeast, it is active, leading to preferential phosphorylation of an approx. 65 kDa protein. A similar pattern of phosphorylation by p51^ferT is seen in mammalian cells.     

The calcium‐sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium‐sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3‐phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H‐89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.     

Activation of a manganese-dependent membrane protein kinase by serine and tyrosine phosphorylation.

A Mn2(+)-dependent serine/threonine protein kinase from rat liver membranes copurifies with the insulin receptor (IR) on wheat germ agglutinin (WGA)-sepharose. The kinase is present in a nonactivated form in membranes but can be activated 20-fold by phosphorylating the WGA-sepharose fraction with casein kinase-1 (CK-1), casein kinase-2 (CK-2), or casein kinase-3 (CK-3). The activated kinase can use IR beta-subunit, myelin basic protein, and histones as substrates. Activation of the kinase seems to proceed by two or more steps. Sodium vanadate and Mn2+ are required in reaction mixtures for activation to be observed, whereas the tyrosine kinase-specific substrate, poly (glu, tyr), completely inhibits activation. These observations suggest that, in addition to serine/threonine phosphorylation by one of the casein kinases, activation of the Mn2(+)-dependent protein kinase also requires tyrosine phosphorylation. Such phosphorylation may be catalyzed by the IR tyrosine kinase.     

Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein

Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton- insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells.     

Interleukin 3-specific tyrosine phosphorylation of a membrane glycoprotein of Mr 150,000 in multi-factor-dependent myeloid cell lines.

Tyrosine phosphorylation of cellular proteins induced by various hematopoietic growth factors such as interleukin 3 (IL3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL4) was studied in several multi-factor-dependent myeloid cell lines. Among the growth factors, IL3 specifically induced rapid tyrosine phosphorylation of a membrane glycoprotein of mol. wt 150 kd (gpp150) in the IL3-dependent cell lines, IC2 and DA-1. The IL3-induced tyrosine phosphorylation of gpp150 was detected within 30 s, reached a maximum at 3 min and decreased thereafter. The concentration of IL3 required for half-maximum stimulation of gpp150 tyrosine phosphorylation with 2.5 x 10(6)/ml cells was approximately 200 pM, which is the same as the dissociation constant for 125I-labeled IL3 binding. gpp150 was constitutively phosphorylated on tyrosine residue(s) in growth factor independent variants, IC2Tr and DA-1Tr, derived from IC2 and DA-1 respectively. Neither variant synthesized IL3. The present findings suggest that tyrosine phosphorylation of gpp150 is a critical event involved in both IL3-dependent and -independent growth.     

The BTB/TAZ domain-containing protein CmBT1-mediated CmANR1 ubiquitination negatively regulates root development in chrysanthemum

Roots are fundamental for plants to adapt to variable environmental conditions.The development of a robust root system is orchestrated by numerous genetic determinants and,among them,the MADS-box gene ANR1 has garnered substantial attention.Prior research has demonstrated that,in chrysanthemum,CmANR1positively regulates root system development.Nevertheless,the upstream regulators involved in the CmANR1-mediated regulation of root development remain unidentified.In this study,we successfully iden...     

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals-possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCdelta with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior.     

PKA phosphorylation of p62/SQSTM1 regulates PB1 domain interaction partner binding

p62, also known as SQSTM1, is a multi-domain signalling scaffold protein involved in numerous critical cellular functions such as autophagy, apoptosis and inflammation. Crucial interactions relevant to these functions are mediated by the N-terminal Phox and Bem1p (PB1) domain, which is divided into two interaction surfaces, one of predominantly acidic and one of basic character. Most known interaction partners, including atypical protein kinase C (aPKC), bind to the basic surface, and acidic–basic interactions at this interface also allow for p62 homopolymerisation. We identify here that the coupling of p62 to the cAMP signalling system is conferred by both the direct binding of cAMP degrading phosphodiesterase-4 (PDE4) to the acidic surface of the p62 PB1 domain and the phosphorylation of the basic surface of this domain by cAMP-dependent protein kinase (PKA). Such phosphorylation is a previously unknown means of regulating PB1 domain interaction partnerships by disrupting the interaction of p62 with basic surface binding partners, such as aPKCs, as well as p62 homopolymerisation. Thus, we uncover a new regulatory mechanism that connects cAMP signalling with the p62 multi-domain signalling scaffold and autophagy cargo receptor protein.