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The focal adhesion adapter protein p130(cas) regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130(cas). AND-34/BCAR3, one of three NSP family members, binds the p130(cas) carboxyl terminus, adjacent to a bipartite p130(cas) Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130(cas). Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130(cas) complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130(cas) to bind the Src SH3 domain through an RPLPSPP motif in the p130(cas) SBD. Although our prior work identified phosphorylation of the serine within the p130(cas) RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130(cas). The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130(cas) complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130(cas) substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130(cas). Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130(cas) and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130(cas) complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130(cas) SBD.

BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas(-/-) murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance., (Copyright © 2011 Elsevier Inc. All rights reserved.)

NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS-PAGE while NSP1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype.

Over-expression of AND-34/BCAR3/NSP2 (BCAR3) or its binding-partner p130Cas/BCAR1 generates anti-estrogen resistance in human breast cancer lines. Here, we have compared BCAR3 to two related homologs, NSP1 and NSP3/CHAT/SHEP, with regards to expression, anti-estrogen resistance, and signaling. BCAR3 is expressed at higher levels in ERalpha-negative, mesenchymal, than in ERalpha-positive, epithelial, breast cancer cell lines. Characterization of "intermediate" epithelial-like cell lines with variable ER-alpha expression reveals that BCAR3 expression correlates with both mesenchymal and ERalpha-negative phenotypes. Levels of the BCAR3/p130Cas complex correlate more strongly with the ERalpha-negative, mesenchymal phenotype than levels of either protein alone. NSP1 and NSP3 are expressed at lower levels than BCAR3 and without correlation to ERalpha/mesenchymal status. Among NSP-transfectants, only BCAR3 transfectants induce anti-estrogen resistance and augment transcription of cyclin D1 promoter constructs. Over-expression of all homologs results in activation of Rac, Cdc42 and Akt, suggesting that these signals are insufficient to induce anti-estrogen resistance. BCAR3 but not NSP1 nor NSP3 transfectants show altered morphology, transitioning from polygonal cell groups to rounded, single cells with numerous blebs. Whereas stable over-expression of BCAR3 in MCF-7 cells does not lead to classic epithelial-to-mesenchymal transition, it does result in down-regulation of cadherin-mediated adhesion and augmentation of fibronectin expression. These studies suggest that BCAR's ability to induce anti-estrogen resistance is greater than that of other NSP homologs and may result from altered interaction of breast cancer cells with each other and the extracellular matrix.

Type 4 cyclic AMP (cAMP) phosphodiesterase (PDE4) inhibitors, a class of compounds in clinical development that activate cAMP-mediated signaling by inhibiting cAMP catabolism, offer a feasible means by which to potentiate glucocorticoid-mediated apoptosis in lymphoid malignancies such as B-cell chronic lymphocytic leukemia (B-CLL). In this study, we show that PDE4 inhibitors up-regulate glucocorticoid receptor (GRalpha) transcript levels in B-CLL cells but not T-CLL cells or Sezary cells or normal circulating T cells, B cells, monocytes, or neutrophils. Because GRalpha transcript half-life does not vary in CLL cells treated with the prototypic PDE4 inhibitor rolipram, the 4-fold increase in GRalpha mRNA levels observed within 4 h of rolipram treatment seems to result from an increase in GRalpha transcription. Rolipram treatment increases levels of transcripts derived from the 1A3 promoter to a greater extent than the 1B promoter. Treatment of B-CLL cells with two other PDE4 inhibitors currently in clinical development also augments GR transcript levels and glucocorticoid-mediated apoptosis. Washout studies show that simultaneous treatment with both drug classes irreversibly augments apoptosis over the same time frame that GR up-regulation occurs. Although treatment of B-CLL cells with glucocorticoids reduces basal GRalpha transcript levels in a dose-related manner, cotreatment with rolipram maintained GRalpha transcript levels above baseline. Our results suggest that as a result of their unusual sensitivity to PDE4 inhibitor-mediated up-regulation of GRalpha expression, treatment of B-CLL patients with combined PDE4 inhibitor/glucocorticoid therapy may be of therapeutic benefit in this disease.

Curcumin, the principle component of the spice turmeric, has been used as an anti-inflammatory medication in India and China for centuries. Recent studies, predominantly using actively dividing cell lines, have suggested that this compound could be used as a chemopreventative or therapeutic agent for epithelial tumors. As curcumin has been reported to inhibit the NIK/IKK complex, an activity that would be expected to induce apoptosis in B cell malignancies, we sought to determine whether curcumin induces apoptosis in vitro in primary chronic lymphocytic leukemia (B-CLL) cells. Primary leukemic cells were incubated with varying dosages of curcumin, followed by assessment for apoptosis. The role of PPARgamma or NF-kappaB signaling in curcumin-induced apoptosis was examined by cotreatment with a PPARgamma antagonist or EMSA of nuclear NFkappaB complexes. We also examined whether a clinically achievable concentration of curcumin (1 microM) would augment the apoptotic effects of fludarabine, dexamethasone, vincristine or the PDE4 inhibitor rolipram. In B-CLL cells from 14 patients, curcumin-induced apoptosis with a mean EC(50) of 5.5 microM. In contrast, the EC(50) for whole mononuclear cells from a healthy donor was 21.8 microM. In a 48 hr wash-out time course, curcumin-induced apoptosis was time-dependent, with a substantial reduction in apoptosis observed when curcumin was removed after 5 hr. Curcumin treatment reduced basal nuclear NF-kappaB levels and 1 microM curcumin augmented both vinca alkaloid and PDE4 inhibitor-induced apoptosis in B-CLL cells. Our studies suggest that curcumin may augment the efficacy of established or experimental therapies for B-CLL.

We report a mechanism by which the adapter protein Gene 33 (also called RALT and MIG6) regulates epidermal growth factor receptor (EGFR) signaling. We find that Gene 33 inhibits EGFR autophosphorylation and specifically blunts epidermal growth factor (EGF)-induced activation and/or phosphorylation of Ras, ERK, JNK, Akt/PKB, and retinoblastoma protein. The Ack homology domain of Gene 33, which contains the previously identified EGFR binding domain, is both necessary and sufficient for this inhibition of EGFR autophosphorylation. The endogenous Gene 33 polypeptide is induced by EGF, platelet-derived growth factor, serum, and dexamethasone (Dex) in Rat 2 rat fibroblasts. Dex induces Gene 33 expression and inhibits EGFR phosphorylation and EGF signaling. RNA interference-mediated silencing of Gene 33 significantly reverses this effect. Overexpression of Gene 33 completely blocks EGF-induced protein and DNA synthesis in Rat 2 cells, whereas gene 33 RNA interference substantially enhances EGF-induced protein and DNA synthesis in Rat 2 cells. Our results indicate that Gene 33 is a physiological feedback inhibitor of the EGFR, functioning to inhibit EGFR phosphorylation and all events induced by EGFR activation. Our results also indicate a role for Gene 33 in the suppression, by Dex, of EGF signaling pathways. We propose that Gene 33 may function in the cross-talk between EGF signaling and other mitogenic and/or stress signaling pathways.

Recently, we found that aquaporin-4 (AQP4) is expressed in the S3 segment of renal proximal tubules of mice but not in rat proximal tubules. Because mice have relatively larger papillae than rats, it was proposed that the renal distribution of AQP4 in various species could be related to their maximum urinary concentrating ability. Therefore, kidneys and other tissues of Merriam's desert kangaroo rat, Dipodomys merriami merriami, which produce extremely concentrated urine (up to 5,000 mosmol/kgH(2)O), were examined for AQP4 expression and localization. Contrary to our expectation, AQP4 immunostaining was undetectable in any region of the kidney, and the absence of AQP4 protein was confirmed by Western blotting. By freeze fracture electron microscopy, orthogonal arrays of intramembraneous particles (OAPs) were not detectable in plasma membranes of principal cells and proximal tubules. However, AQP4 protein was readily detectable in gastric parietal and brain astroglial cells. Northern blotting failed to detect AQP4 mRNA in kangaroo rat kidneys, whereas both in situ hybridization and RT-PCR experiments did reveal AQP4 mRNA in collecting ducts and proximal tubules of the S3 segment. These results suggest that renal expression of AQP4 in the kangaroo rat kidney is regulated at the transcriptional or translational level, and the absence of AQP4 may be critical for the extreme urinary concentration that occurs in this species.

Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extracellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH(2)-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences.

The amino acid sequences of two tryptic peptides derived from purified preparations of I1PP2A indicated that this potent heat-stable protein inhibitor of protein phosphatase 2A (PP2A) may be equivalent to putative histocompatibility leukocyte antigens class II-associated protein I (PHAP-I). Experiments using purified preparations of recombinant human PHAP-I confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 4 nM PHAP-I, similar to the half-maximal inhibition obtained with purified preparations of bovine kidney I1PP2A. In addition, PHAP-I did not affect the activities of protein phosphatase 1, 2B, and 2C in a manner analogous to that of I1PP2A. Together, the results establish the identity of I1PP2A on a firm basis.

Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor proteins designated I1PP2A and I2PP2A have been purified to apparent homogeneity from extracts of bovine kidney (Li, M., Guo, H., and Damuni, Z. (1995) Biochemistry 34, 1988-1996). N-terminal and internal amino acid sequencing indicated that I2PP2A was a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia. Experiments using purified preparations of recombinant human SET confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 2 nM SET. By contrast, SET (up to 20 nM) did not affect the activities of purified preparations of protein phosphatases 1, 2B, and 2C. The results indicate that SET is a potent and specific inhibitor of PP2A and suggest that impaired regulation of PP2A may contribute to acute myeloid leukemogenesis.

Insulin-stimulated protamine kinase (cPK) and protein kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent Km for eIF-4E was about 1.2 and 50 microM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and < or = 5% activity with eIF-4ES209A and eIF-4ET210A, respectively, and eIF-4ES209A was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in m7GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser209 and Thr210, that the hydroxyl group or phosphorylation of Thr210 is necessary for cPK to act on Ser209, and that Ser209 phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.

More than 15 subunits of the integrin family of cell surface adhesion molecules have been identified. The alpha 6 beta 4 integrin has recently been identified as a component of hemidesmosomes of stratified squamous epithelium. The monoclonal antibody (mAb) 346-11A binds to the beta 4 subunit in mice. Sequence analysis of a cDNA clone coding for this epitope localizes reaction of the mAb to a portion about half way through the extracellular domain at the beginning of the cysteine-rich region. Sites of beta 4 expression in mice were detected by autoradiographic analysis of tissues collected from mice 24 or 48 h after intravenous injection of 125I-labeled mAb 346-11A. This non-quantitative technique emphasizes detection of antigen exposed in the vascular space. These data show that in addition to the epithelia of several organs, the endothelia of intermediate vessels throughout the body are sites of beta 4 expression. In particular, endothelium of larger vessels but not capillaries in lung, vessels in thymus, spleen and Peyer's patches, and portal vessels but not the central veins of the liver, are positive. The expression of the beta 4 integrin in blood vessels may indicate a specialized function for beta 4 at these sites that is distinct from its role in hemidesmosome-mediated attachment.

Gene 33 is a multihormonally regulated rat gene whose transcription is rapidly and markedly enhanced by glucocorticoids, insulin, or cAMP. A cDNA clone (p216) containing a nearly full-length DNA complementary to the mRNA of this gene was isolated from a cDNA library and sequenced. The cDNA represents the entire mRNA transcript, except for three bases at the 5' terminus. The message is 2970 nucleotides long and can encode a protein of 459 amino acids with a molecular mass of 49,919 Da. The deduced amino acid sequence is rich in proline and serine but bears little homology to any known sequences. The 5' untranslated region, rich in G + C, is 270 nucleotides long and contains two apparently nonfunctional AUG initiator codons. The 3' untranslated sequence, rich in A + U, is 1323 nucleotides long and has a polyadenylation signal 13 bases upstream of the poly(A) tract. Two mRNA transcripts of the gene, which appear to be the consequence of alternative usage of splice sites, have been identified. The less abundant mRNA is truncated by 228 nucleotides in the protein coding region. The reading frame is maintained and this mRNA can code for a protein of 383 amino acids with a calculated molecular mass of 42,204 Da.