A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the b 2 -adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor±scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold±receptor interactions. Gravin constructs lacking the AKAP domain displayed no binding to the receptor. Metabolic labeling studies in vivo demonstrate agonist-stimulated phosphorylation of gravin and enhanced gravin±receptor association. Analysis of the AKAP domain revealed two canonical PKA sites phosphorylated in response to elevated cAMP, blocked by PKA inhibitor, and essential for scaffold±receptor association and for resensitization of the receptor. The AKAP appears to provide the catalytic PKA activity responsible for phosphorylation of the scaffold in response to agonist activation of the receptor as well as for the association of the scaffold with the receptor, a step critical to receptor resensitization. Keywords: b-adrenergic receptor/AKAP/gravin/protein kinase A/protein kinase C/scaffold IntroductionRecent advances in our understanding of the workings of cell signaling have highlighted the roles of molecules, often referred to as`scaffolds', in organizing the interactions of molecules in signaling pathways. In Saccharomyces cerevisiae, for example, STE5 is an essential component of the pheromone-mediated mitogen-activated protein kinase (MAPK) pathway. The STE5 scaffold recruits MAPK cascade members STE11, STE7 and FUS3 to generate speci®city for the pheromone pathway (Kim et al., 1998). In vertebrates, A-kinase-anchoring proteins (AKAPs) constitute a diverse family of such scaffold molecules, organizing not only protein kinases, but also protein phosphatases and other molecules, into multivalent signaling complexes (Pawson and Scott, 1997;Colledge and Scott, 1999;Bauman and Scott, 2002). AKAPs bind the RII subunits of the cAMP-dependent kinase (PKA), providing discrete localization of PKA within the cell.The muscle-speci®c AKAP, termed`mAKAP', coordinates multivalent signaling complexes in both the sarcoplasmic reticulum and perinuclear membrane of muscle cells . Another family of AKAPs are derived from a single gene, producing the splice products AKAP350, AKAP450, CG-NAP and Yotiao that are involved in coordinating multivaltent signaling complexes with N-methyl-D-aspartate (NMDA) receptors, protein phosphatases PP1 and calcineurin, as well as PKA Bauman and Scott, 2002). Gravin (also known as AKAP250 and AKAP12) is another prominent member of the AKAP family, a family that includes >50 members. As scaffold molecules, the AKAPs display binding sites for several well known protein kinases and phosphatases. Gravin, for example, binds the RII subunit of PKA, protein kinase C (PKC), protein phosphatase 2B (PP2B) (Nauert et al., 1997;Shih et al., 1999;Fan et al.,...

The novel Bruton's tyrosine kinase inhibitor ibrutinib has demonstrated high response rates in B-cell lymphomas; however, a growing number of ibrutinib-treated patients relapse with resistance and fulminant progression. Using chemical proteomics and an organotypic cell-based drug screening assay, we determine the functional role of the tumour microenvironment (TME) in ibrutinib activity and acquired ibrutinib resistance. We demonstrate that MCL cells develop ibrutinib resistance through evolutionary processes driven by dynamic feedback between MCL cells and TME, leading to kinome adaptive reprogramming, bypassing the effect of ibrutinib and reciprocal activation of PI3K-AKT-mTOR and integrin-β1 signalling. Combinatorial disruption of B-cell receptor signalling and PI3K-AKT-mTOR axis leads to release of MCL cells from TME, reversal of drug resistance and enhanced anti-MCL activity in MCL patient samples and patient-derived xenograft models. This study unifies TME-mediated de novo and acquired drug resistance mechanisms and provides a novel combination therapeutic strategy against MCL and other B-cell malignancies.

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype and proliferate and synthesize fibrosis components. Endothelin-1 (ET-1), which inhibited the growth of human myofibroblastic HSC, increased the formation of two NF-B DNA binding complexes; this effect was also observed with tumor necrosis factor-␣ (TNF-␣). The complexes were identified as the p50/p50 and p50/p65 NF-B dimers. Activation of NF-B was associated with the degradation of the inhibitory protein IB-␣; no IB-␤ was detected. Activation of NF-B and degradation of IB-␣ were prevented by the NF-B inhibitors sodium salicylate and MG-132. In addition to cyclooxygenase-1 (COX-1), COX-2 is also constitutively expressed in human HSC, and the use of dexamethasone and of SC-58125, a selective COX-2 inhibitor, revealed that COX-2 accounts for basal COX activity. Moreover, COX-2 mRNA and protein were up-regulated by ET-1 and TNF-␣, whereas COX-1 was unaffected. Induction of COX-2 and stimulation of COX activity by ET-1 and TNF-␣ were prevented by sodium salicylate and MG-132, suggesting that activation of NF-B by either factor is needed for stimulation of COX-2. Finally, SC-58125 and dexamethasone reduced the growth inhibitory effect of ET-1 and TNF-␣, indicating that activation of COX-2 is required for inhibition of HSC proliferation. Taken together, our results suggest that NF-B, by inducing COX-2 expression, may play an important role in the negative regulation of human myofibroblastic HSC proliferation.

Proliferation of hepatic myofibroblasts (hMF) is central for the development of fibrosis during liver injury, and factors that may limit their growth are potential antifibrotic agents. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with growth-regulating properties, either via Edg receptors or through intracellular actions. In this study, we examined the effects of S1P on the proliferation of human hMF. Human hMF expressed mRNAs for the S1P receptors Edg1, Edg3, and Edg5. These receptors were functional at nanomolar concentrations and coupled to pertussis toxin-sensitive and -insensitive G proteins, as demonstrated in guanosine 5-3-O-(thio)triphosphate binding assays. S1P potently inhibited hMF growth (IC 50 ‫؍‬ 1 M), in a pertussis toxininsensitive manner. Analysis of the mechanisms involved in growth inhibition revealed that S1P rapidly increased prostaglandin E 2 production and in turn cAMP, two growth inhibitory messengers for hMF; C 2 -ceramide and sphingosine, which inhibited hMF proliferation, did not affect cAMP levels. Production of cAMP by S1P was abolished by NS-398, a selective inhibitor of COX-2. Also, S1P potently induced COX-2 protein expression. Blocking COX-2 by NS-398 blunted the antiproliferative effect of S1P. We conclude that S1P inhibits proliferation of hMF, probably via an intracellular mechanism, through early COX-2-dependent release of prostaglandin E 2 and cAMP, and delayed COX-2 induction. Our results shed light on a novel role for S1P as a growth inhibitory mediator and point out its potential involvement in the negative regulation of liver fibrogenesis.

Proliferation of myofibroblastic hepatic stellate cells (HSC) in response to growth factors is essential for the development of liver fibrosis. We have reported that prostaglandins (PG) and cyclic AMP (cAMP) inhibit growth of human HSC. This PG/cAMP pathway transduces the endothelin (ET) B-mediated antiproliferative effect of endothelin-1 (ET-1) and up-regulates ETB receptors. Here, we show that platelet-derived growth factor (PDGF)-BB and thrombin, although mitogenic, generate growth inhibitory PGE 2 in myofibroblastic human HSC. The two peptides elicit early PGE 2 and cAMP synthesis, and also promote delayed induction of cyclooxygenase (COX)-2. Both early and delayed production of PGE 2 counteract the mitogenic effect of PDGF-BB and thrombin because: (i) pretreatment with the COX inhibitor ibuprofen markedly enhances the mitogenic effect of both peptides; (ii) blocking early synthesis of PGE 2 greatly enhances extracellular signal-regulated kinase (ERK) activation by both growth factors; (iii) enhancement of DNA synthesis by ibuprofen is only lost when the inhibitor is added after COX-2 induction has occurred. Finally, PDGF-BB and thrombin raise ETB receptors through the PG pathway. Thus, ibuprofen blunts growth factor-induced increase in ETB receptors. Up-regulation of the growth inhibitory ETB receptors by both mitogens may enhance the antiproliferative effect of ET-1 and thereby establish a negative feedback of their mitogenic effect. Our results shed light on novel growth inhibitory signals evoked by two mitogenic growth factors expressed during liver injury.