Neuroblastoma, an embryonal tumor of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer1. High-risk neuroblastomas, prevalent in the majority of patients, are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal2,3. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germline. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK cDNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed IL-3-dependent murine hematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to a small-molecule inhibitor of ALK, TAE6844. Furthermore, two human neuroblastoma cell lines harboring the F1174L mutation were sensitive to the inhibitor. Cytotoxicity was associated with increased levels of apoptosis as measured by TUNEL-labeling. shRNA-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumors and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

Recoverin, a recently discovered member of the EF-hand superfamily of Ca2+-bindig proteins, serves as a Ca2+ sensor in vision. The amino terminus of the protein from retinal rod cells contains a covalently attached myristoyl or related N-acyl group. We report here studies of unmyrtylated and myristoylated recombinant recoverin designed to delineate the biological role of this hydrophobic unit. Ca2+ induces the binding of both the unmyristoylated and myristoylated proteins to phenyl-agarose, a hydrophobic support. MATERIALS AND METHODSRecoverin. Recombinant myristoylated and unmyristoylated recoverins were expressed in the overproducing E. coli strains pTrec2/pBB131/DH5aF' and pTrec2/DH5a, respectively, and were purified by a modification of a previously described procedure (5, 15). Cells from 20 liters of a bacterial culture were resuspended in 200 ml of 100 mM KCl/1 mM dithiothreitol/1 mM MgCl2/50 mM potassium Hepes, pH 7.5 (buffer A) supplied with 1 mM EGTA and disrupted by sonication. Streptomycin sulfate was added with stirring over a 10-min period to a final concentration of 0.1% from a 5% stock prepared in buffer A, and the cell debris was removed by centrifugation at 40C for 30 min at 18,000 x g. The cleared lysate was filtered through filter paper (Whatman 1), brought to 2 mM CaCl2, and applied to an 80-ml phenyl-Sepharose CL-4B (Pharmacia) column previously equilibrated with buffer A containing 2 mM CaC12. The column was washed with 400 ml of the same buffer, and the fraction containing recoverin was eluted with 100-150 ml of buffer A containing 2 mM EGTA. The eluate was diluted with 3 volumes of cold water, applied to a HiLoad 26/10 Q-Sepharose HP column (Pharmacia), and chromatographed with a gradient of 0-200 mM KCl in 500 ml of 20 mM Tris-HCl, pH 8.0/1 mM dithiothreitol/1 mM MgCl2/0.1 mM EGTA. Purified recoverin was concentrated to 10-20 mg/ml using Centriprep 10 centrifugal concentrators (Amicon), flash-frozen in liquid nitrogen, and stored at -70'C.To produce tritium-labeled myristoylated recoverin, the overproducing strain pTrec2/pBB131/DHSFa' containing the yeast N-myristoyltransferase expression vector (22) 11569The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Recoverin, a recently discovered member of the EF hand superfamily, serves as a calcium sensor in vision. We report here the crystal structure of recombinant unmyristoylated recoverin at 1.9 A resolution. The four EF hands of the protein are arranged in a compact array that contrasts with the dumbbell shape of calmodulin and troponin C. A calcium ion is bound to EF hand 3, while EF hand 2 can bind samarium but not calcium in this crystal form. The other two EF hands have novel structural features that prevent or impair calcium binding. A concave hydrophobic surface formed by EF hands 1 and 2 may participate in the read out of calcium signals by recoverin and its homologs.

The receptor tyrosine kinase EphB4 and its ligand EphrinB2 play a crucial role in vascular development during embryogenesis. The soluble monomeric derivative of the extracellular domain of EphB4 (sEphB4) was designed as an antagonist of EphB4/EphrinB2 signaling. sEphB4 blocks activation of EphB4 and EphrinB2; suppresses endothelial cell migration, adhesion, and tube formation in vitro; and inhibits the angiogenic effects of various growth factors (VEGF and bFGF) in vivo. sEphB4 also inhibits tumor growth in murine tumor xenograft models. sEphB4 IntroductionDifferentiation of mesodermal cells to angioblasts occurs with simultaneous commitment to either arterial or venous lineage. Angioblasts spontaneously aggregate, proliferate, and differentiate to form endothelial tubes of each lineage. Independently developing arterial and venous vascular networks eventually join to form the original cardiovascular loop in the process of vasculogenesis. [1][2][3] Sprouting of new vessels from this primary complex, or angiogenesis, is mediated by growth factors that induce endothelial cell (EC) proliferation, migration, and assembly, followed closely by the recruitment of perivascular cells, including smooth muscle cells, and remodeling of the extracellular matrix. 4,5 A number of ECspecific receptor tyrosine kinases have been identified that play important roles in the early development of blood vessels and formation of the cardiovascular system, and include VEGF receptors and Tie-1 and Tie-2 receptors. [5][6][7][8][9][10][11] More recently, Eph receptors and their ligands have been shown to play a critical role in the development and maturation of the cardiovascular system. [11][12][13] The Ephs and Ephrins together comprise the largest of the receptor tyrosine kinase subfamilies (with 14 receptors and 8 ligands) and are subdivided into EphA and EphB categories based on sequence homologies and binding properties to Ephrin ligands. EphA receptors bind to glycosylphosphatidylinositol (GPI)-anchored Ephrin ligands (EphrinA subfamily), whereas EphB receptors bind Ephrin ligands that contain transmembrane and cytoplasmic domains (EphrinB subfamily). 14 The extracellular domain of Eph receptors consists of a ligand-binding (globular or G) and a cysteine-rich (C) domain followed by 2 fibronectin III-like repeats (F1 and F2). The intracellular domain contains an autoinhibitory tyrosine in the juxtamembrane region, followed by a kinase domain, sterile ␣ and PDZ-binding motifs. 15,16 Eph receptor tyrosine kinases and their Ephrin ligands regulate a diverse array of cellular functions such as cell migration, repulsion, and adhesion, but lack effects on cell proliferation. 9,[17][18][19][20][21] These functions are dependent on bidirectional signals between cells expressing receptors and cells expressing ligands, which, for uniformity of communication, are termed "forward" and "reverse" signaling, respectively. 6,9,11,[21][22][23][24][25] EphrinB2 is specifically expressed in arterial angioblasts and endothelial and perivascular mesench...

Precise spatial and temporal regulation of Rho GTPases is required in controlling F-actin-based changes in cell morphology. The molecular mechanisms through which microtubules (MTs) modulate the activity of RhoGTPases and regulate the actin cytoskeleton are unclear. Here we show that p21-activated-kinase 4 (PAK4) mediates morphological changes through its association with the Rho-family guanine nucleotide exchange factor (GEF), GEF-H1. We show that this association is dependent upon a novel GEF-H1 interaction domain (GID) within PAK4. Further, we show that PAK4-mediated phosphorylation of Ser810 acts as a switch to block GEF-H1-dependent stress fiber formation while promoting the formation of lamellipodia in NIH-3T3 cells. We found that the endogenous PAK4-GEF-H1 complex associates with MTs and that PAK4 phosphorylation of MT-bound GEF-H1 releases it into the cytoplasm of NIH-3T3 cells, which coincides with the dissolution of stress fibers. Our observations propose a novel role for PAK4 in GEF-H1-dependent crosstalk between MTs and the actin cytoskeleton.