Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor B (NF-B). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-B pathway, which mediates NGF survival and differentiation responses.

Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G 1 . Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G 0 /G 1 . Here we show that RACK1 overexpression induces a partial G 1 arrest by suppressing Src activity at the G 1 checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G 1 /S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G 1 /S progression. RACK1 suppresses Src-but not mitogen-activated protein kinasedependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G 1 that works via an endogenous inhibitor of the Src kinase.

Previously, we showed that Src tyrosine kinases are activated early in the development of human colon cancer and are suppressed as intestinal cells differentiate. We identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src. Here we show (by overexpressing RACK1, depleting Src or RACK1 and utilizing cell-permeable peptides that perturb RACK1's interaction with Src) that RACK1 regulates growth of colon cells by suppressing Src activity at G 1 and mitotic checkpoints, and consequently delaying cell cycle progression. Activated Src rescues RACK1-inhibited growth of HT-29 cells. Conversely, inhibiting Src abolishes growth promoted by RACK1 depletion in normal cells. Two potential mechanisms whereby RACK1 regulates mitotic exit are identified: suppression of Src-mediated Sam68 phosphorylation and maintenance of the cyclin-dependent kinase (CDK) 1-cyclin B complex in an active state. Our results reveal novel mechanisms of cell cycle control in G 1 and mitosis of colon cells. The significance of this work lies in the discovery of a mechanism by which the growth of colon cancer cells can be slowed, by RACK1 suppression of an oncogenic kinase at critical cell cycle checkpoints. Small molecules that mimic RACK1 function may provide a powerful new approach to the treatment of colon cancer.

The neurotrophin nerve growth factor (NGF) supports neuronal survival by activating the transcription factor nuclear factor-B (NF-B). We report here, for the first time, the identification of p75-associated kinase that mediates NGF-driven NF-B activation. Using coimmunoprecipitation, we demonstrate an NGF-dependent association of interleukin 1 receptor-associated kinase (IRAK) with the p75 neurotrophin receptor in PC12 cells. Our results reveal that IRAK is recruited to the p75-NGF receptor leading to formation of a complex between IRAK, atypical protein kinase C interacting protein, p62, and TRAF6. Activation of NF-B occurs predominantly through the p75 receptor, and TrkA activity suppresses NF-B activation and retards IB␤ degradation. In addition, we observe a requirement for the kinase activity of IRAK in mediating NGF-induced NF-B activation, recruitment of the adapter protein p62 to the p75 receptor, and cell survival. Moreover, p75-IRAK-mediated B activation and the recruitment of IKK␤, but not IKK␣, to the receptor require p62. Altogether, our data provide novel information regarding the proximal components involved in p75 receptor signaling and underscore the importance of the atypical PKC interacting protein p62 in this process. The transcription factor, nuclear factor-B (NF-B)1 regulates the expression of a wide variety of genes involved in immunity, inflammation, apoptosis, and other cellular processes (1-3). NF-B resides in the cytoplasm in an inactive form bound to an inhibitory protein of the IB family. Activation of NF-B by an external stimulus involves phosphorylation and rapid degradation of IB proteins by the IB kinase (IKK) complex, leading to nuclear translocation of NF-B. The proinflammatory cytokines IL-1␤ and TNF␣ are the most well characterized stimuli that lead to the activation of NF-B.Binding of interleukin-1 (IL-1) to its receptor promotes the association of IL-1 receptor-associated kinase (IRAK) with the receptor (4), through the adapter protein MyD88 (5). IRAK then gets highly phosphorylated and leaves the receptor complex to interact with TRAF6, a member of the TNF receptor associated factor family (6). The IRAK⅐TRAF6 interaction triggers kinase cascades that lead to the activation of NF-B (6 -8).IRAK is a serine/threonine-specific protein kinase that shares 25% sequence identity with human mixed-lineage kinase, 30 -33% sequence identity with a protein kinase that is a product of the pto gene of tomato plant, and 32% identity to the kinase domain of Drosophila pelle, which is involved in the activation of Dorsal, the Drosophila equivalent of NF-B (4). These kinases define a subgroup of cytoplasmic kinases called the serine/threonine innate immunity kinases (SIIK) group (9). IRAK is a multidomain protein containing an N-terminal death domain of 120 amino acids, a central 300-amino acid kinase domain, and an undetermined C-terminal domain that is absent in pelle (10). Two other IRAK-related proteins IRAK-2 and IRAK-M (for its higher levels of expression in cells of monocytic lineage)...

Several recent reports support a dual role of p75 NTR in cell death, as well as survival, depending on the physiological or developmental stage of the cells. Coexpression of the TrkA receptor with p75 NTR further enhances the complexity of nerve growth factor (NGF) signaling. Recent identification of serine/threonine kinases that interact with the p75 NTR provides an explanation for the lack of an apparent kinase domain needed for signaling. In this report, we review the possible roles of the intracellular proteins that directly interact with the p75 NTR , atypical protein kinase C (PKC) binding protein, p62 and second messengers in the functional antagonism exhibited by TrkA and p75 NTR with an emphasis on the nuclear factor-kappa B activation pathway.

SummaryWe present data from a phase II study investigating a novel treatment strategy for relapsed/refractory mantle cell lymphoma (MCL). Twenty-six patients received lenalidomide 25 mg/d (days 1-21 of a 28-d cycle) for up to 6 cycles followed by low-dose maintenance lenalidomide (15 mg) in responding patients. Eight patients achieved complete or partial response to give an overall response rate of 31% with median response duration of 22Á2 months [95% confidence interval (CI) 0Á0-53Á6] and median progression-free survival (PFS) of 3Á9 months (95% CI 0Á0-11Á1). An additional six patients (23%) achieved stable disease. Eleven patients received maintenance with median PFS of 14Á6 months (95% CI 7Á3-21Á9). Correlative studies showed that peripheral T and Natural Killer (NK) cells increased in responding patients by 40-60% over the first 6 cycles with an initial dip in NK cells suggestive of tumour infiltration. Peripheral regulatory T cells were increased in MCL patients (P = 0Á001) and expanded further following lenalidomide. Sequential plasma analysis showed increased IL12 p40 and IL7 alongside decreased MMP9, IL10, and adiponectin. Finally, a significant correlation (P = 0Á02) between gender and response suggested that female MCL patients were more sensitive to lenalidomide than males. In summary, we confirm the activity, safety and immunomodulatory properties of lenalidomide in MCL and highlight its potential as a low-dose maintenance agent.

Earlier we showed that RACK1 regulates growth of human colon cells by suppressing Src activity at G 1 and mitotic checkpoints. Here, we show that RACK1 also induces apoptosis of the cells, partly by inhibiting Src. In the intrinsic pathway, RACK1 inhibits expression of anti-apoptotic Bcl-2 and Bcl-X L , induces expression of proapoptotic Bim, targets Bim and Bax to the mitochondria, induces oligomerization of Bax (which requires Bim and inhibition of Src), depolarizes mitochondria membranes, releases cytochrome c, and activates caspases-9 and -3 and death substrates. Bax and Bim are required for RACK1-mediated mitochondrial cell death. RACK1-induced oligomerization of Bax is required for staurosporine-mediated cell death. RACK1 also induces apoptosis by blocking Src activation of the Akt cell survival pathway. This leads to activation of the transcription factor FOXO3, a potent inducer of apoptosis and G 1 arrest. Collectively, our results show that RACK1, partly by inhibiting Src, promotes mitochondrial cell death and blocks Akt-mediated cell survival. Thus, RACK1 inhibits growth and induces death of colon cells. Exploitation of these dual functions could lead to novel colon cancer therapies that mimic RACK1 function.

Summary Inhibition of histone deacetylase 6 (HDAC6)-dependent aggresome function by pan HDAC inhibitors was recently reported to be a key mechanism underlying the synergistic activity between proteasome inhibitors and HDAC inhibitors in a variety of tumour types. Because these combinations induce significant thrombocytopenia in vivo, we examined whether less toxic, isotype-selective HDAC inhibitors may still synergize with proteasome inhibitors, and if so, by what mechanisms. Here, we showed that the class I HDAC inhibitor, MGCD0103, has a potent antiproliferative activity in Hodgkin lymphoma (HL) cell lines. Furthermore, MGCD0103 induced tumour necrosis factor α (TNF-α) expression and secretion, which was associated with nuclear factor (NF)-κB activation. Selective inhibition of TNF- α expression by short interfering mRNA, or inhibition of MGCD0103-induced NF-kB activation by proteasome inhibitors enhanced MGCD0103-induced cell death. Thus, our results demonstrate that MGCD0103 may synergize with proteasome inhibitors by HDAC6-independent mechanisms, providing mechanistic rationale for exploring this potentially less toxic combination for the treatment of lymphoma.