The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3) downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here, we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is essential for Ca(2+) influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca(2+) influx and TNF-induced necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis.

Here we show that p53 protein is physically associated with tubulin in vivo and in vitro, and that it localizes to cellular microtubules. Treatment with vincristine or paclitaxel before DNA-damage or before leptomycin B treatment reduces nuclear accumulation of p53 and expression of mdm2 and p21. Overexpression of dynamitin or microinjection of anti-dynein antibody before DNA damage abrogates nuclear accumulation of p53. Our results indicate that transport of p53 along microtubules is dynein-dependent. The first 25 amino acids of p53 contain the residues that are essential for binding to microtubules. We propose that functional microtubules and the dynein motor protein participate in transport of p53 and facilitate its accumulation in the nucleus after DNA damage.

Intracellular signalling following mitogenic stimulation of quiescent cells involves the initiation of a phosphorylation cascade that leads to the rapid and reversible activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2. MAP kinase activation is mediated by dual phosphorylation within the motif Thr-Glu-Tyr by MAP kinase kinase (MEK). Following activation, the MAP kinases translocate into the nucleus where they phosphorylate several transduction targets, including transcription factors. We have previously identified PAC1 as an immediate-early mitogen-inducible tyrosine phosphatase in nuclei of T cells. Here we present several lines of evidence indicating that PAC1 is a physiologically relevant MAP kinase phosphatase. Recombinant PAC1 in vitro is a dual-specific Thr/Tyr phosphatase with stringent substrate specificity for MAP kinase. Constitutive expression of PAC1 in vivo leads to inhibition of MAP kinase activity normally stimulated by epidermal growth factor, phorbol myristyl acetate, or T-cell receptor crosslinking. The inactivation of MAP kinase by PAC1 results in inhibition of MAP kinase-regulated reporter gene expression.

CD97, a membrane protein expressed at high levels on inflammatory cells and some carcinomas, is a member of the adhesion G protein-coupled receptor family, whose members have bipartite structures consisting of an extracellular peptide containing adhesion motifs noncovalently coupled to a class B 7-transmembrane domain. CD97␣, the extracellular domain of CD97, contains 3 to 5 fibrillin class 1 epidermal growth factor (EGF)-like repeats, an Arg-Gly-Asp (RGD) tripeptide, and a mucin stalk. We show here that CD97␣ promotes angiogenesis in vivo as demonstrated with purified protein in a directed in vivo angiogenesis assay (DIVAA) and by enhanced vascularization of developing tumors expressing CD97. These data suggest that CD97 can contribute to angiogenesis associated with inflammation and tumor progression. Strong integrin ␣5␤1 interactions with CD97 have been identified, but ␣v␤3 also contributes to cell attachment. Furthermore, soluble CD97 acts as a potent chemoattractant for migration and invasion of human umbilical vein endothelial cells (HUVECs), and this function is integrin dependent. CD97 EGF-like repeat 4 is known to bind chondroitin sulfate. It was found that coengagement of ␣5␤1 and chondroitotin sulfate proteoglycan by CD97 synergistically initiates endothelial cell invasion. Integrin ␣5␤1 is the first high-affinity cellular counterreceptor that has been identified for a member within this family of adhesion receptors. ( IntroductionThe adhesion G protein-coupled receptors are membrane-bound proteins with long N-termini containing multiple and distinct combinations of adhesion motifs. 1 These proteins contain domains such as epidermal growth factor (EGF)-like, cadherin, lectin, laminin, olfactomedin, immunoglobulin, or thrombospondin modules. The extracellular domains are bound to a conserved family of 7 transmembrane (TM) receptors that are distantly related to the class B, or secretin family, G protein-coupled receptors. A characteristic feature of the receptors within this family is their posttranslational proteolysis prior to expression at the cell membrane from a single peptide to 2 noncovalently associated subunits. [2][3][4] The ␣ subunit comprises the majority of the extracellular domain and contains the adhesion motifs, whereas the ␤ subunit comprises the 7 TM receptor with a short extracellular extension. A lack of knowledge concerning high-affinity ligand or counterreceptor interactions has impeded progress in defining signaling pathways and physiologic actions of this family of receptors.CD97 belongs to the EGF-TM7 subgroup of adhesion G proteincoupled receptors. 5 The proteins in this group contain varying numbers of extracellular fibrillin class 1 type EGF-like repeats and include CD97, EMR1, EMR2, EMR3, and EMR4. CD97 is found in myeloid cells, lymphoid cells, and muscle cells, whereas the other members of this family are predominantly in myeloid lineage cells. 3,[6][7][8] In addition, CD97 is often expressed in advanced-stage thyroid, colorectal, gastric, esophageal, and pancreatic carc...

CD97, an adhesion-linked G-protein-coupled receptor (GPCR), is induced in multiple epithelial cancer lineages. We address here the signaling properties and the functional significance of CD97 expression in prostate cancer. Our findings show that CD97 signals through Ga12/13 to increase RHO-GTP levels. CD97 functioned to mediate invasion in prostate cancer cells, at least in part, by associating with lysophosphatidic acid receptor 1 (LPAR1), leading to enhanced LPA-dependent RHO and extracellular signal-regulated kinase activation. Consistent with its role in invasion, depletion of CD97 in PC3 cells resulted in decreased bone metastasis without affecting subcutaneous tumor growth. Furthermore, CD97 heterodimerized and functionally synergized with LPAR1, a GPCR implicated in cancer progression. We also found that CD97 and LPAR expression were significantly correlated in clinical prostate cancer specimens. Taken together, these findings support the investigation of CD97 as a potential therapeutic cancer target. Cancer Res; 71(23); 7301-11. Ó2011 AACR.

The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins are important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the Rad, Gem, and Kir family) for which biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) α and β. Gem binds ROKβ independently of RhoA in the ROKβ coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKβ-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKβ. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKβ- and Rad opposed ROKα-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKβ containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKβ is required for the effects observed. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK.

Sonic hedgehog (Shh) signaling regulates both digit number and identity,but how different distinct digit types (identities) are specified remains unclear. Shh regulates digit formation largely by preventing cleavage of the Gli3 transcription factor to a repressor form that shuts off expression of Shh target genes. The functionally redundant 5′Hoxd genes regulate digit pattern downstream of Shh and Gli3, through as yet unknown targets. Enforced expression of any of several 5′Hoxd genes causes polydactyly of different distinct digit types with posterior transformations in a Gli3(+) background, whereas, in Gli3 null limbs,polydactylous digits are all similar, short and dysmorphic, even though endogenous 5′Hoxd genes are broadly misexpressed. We show that Hoxd12 interacts genetically and physically with Gli3, and can convert the Gli3 repressor into an activator of Shh target genes. Several 5′Hoxd genes,expressed differentially across the limb bud, interact physically with Gli3. We propose that a varying [Gli3]:[total Hoxd] ratio across the limb bud leads to differential activation of Gli3 target genes and contributes to the regulation of digit pattern. The resulting altered balance between `effective'Gli3 activating and repressing functions may also serve to extend the Shh activity gradient spatially or temporally.

Gem is a small GTP-binding protein that has a ras-like core and extended chains at each terminus. The primary structure of Gem and other RGK family members (Rad, Rem, and Rem2) predicts a GTPase deficiency, leading to the question of how Gem functional activity is regulated. Two functions for Gem have been demonstrated, including inhibition of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. These functions for Gem have been ascribed to its interaction with the calcium channel ␤ subunit and Rho kinase ␤, respectively. We show here that these functions are separable and regulated by distinct structural modifications to Gem. Phosphorylation of serines 261 and 289, located in the C-terminal extension, is required for Gem-mediated cytoskeletal reorganization, while GTP and possibly calmodulin binding are required for calcium channel inhibition. In addition to regulating cytoskeletal reorganization, phosphorylation of serine 289 in conjunction with serine 23 results in bidentate 14-3-3 binding, leading to increased Gem protein half-life. Evidence presented shows that phosphorylation of serine 261 is mediated via a cdc42/protein kinase Cdependent pathway. These data demonstrate that phosphorylation of serines 261 and 289, outside the GTPbinding region of Gem, controls its inhibition of Rho kinase ␤ and associated changes in the cytoskeleton.