Activation of the ligand binding function of integrin heterodimers requires transmission of an "inside-out" signal from their small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alpha(IIb)beta(3) has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the beta cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin that are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the beta cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation.

Abbreviations used in this paper: CT, cytoplasmic tail; HUVEC, human umbilical vein endothelial cell; talin-H, talin head domain.The online version of this paper contains supplemental material. IntroductionIntegrin activation, the rapid transition from a low to a high affi nity state for ligand, regulates the numerous cellular responses consequent to integrin engagement by extracellular matrix proteins or counter-receptors on other cells ( Hynes, 2002 ). This transformation is tightly controlled by the integrin cytoplasmic tails (CTs) ( Qin et al., 2004 ;Ma et al., 2007 ). Mutational and structural analyses suggest that the ␤ 3 CT can be divided two regions, and both infl uence integrin activation. The membraneproximal region of the ␤ 3 CT is primarily ␣ -helix, which interacts with the membrane-proximal helix of the ␣ subunit through several electrostatic and hydrophobic bonds ( Vinogradova et al., 2002 ). Unclasping of the complex is a critical event in integrin activation ( Hughes et al., 1996 ;Kim et al., 2003 ;Ma et al., 2006 ). The membrane-distal region of the ␤ 3 CT contains two NXXY turn motifs, NPLY 747 and NITY 759 , which are separated by a short helix containing a T/S cluster, the TS 752 T region ( Fig. 1 A ). The head domain of talin (talin-H) docks at the NPLY 747 motif through its F 3 domain and also interacts with the membrane-proximal region, perturbing the membrane clasp and leading to at least partial integrin activation ( Vinogradova et al., 2002 ;Tadokoro et al., 2003 ;Wegener et al., 2007 ). The T/S cluster and the NITY motif are also critical for integrin activation ( Chen et al., 1994 ;O ' Toole et al., 1995 ;Xi et al., 2003 ;Ma et al., 2006 ). However, the mechanisms underlying their effects remain unresolved. In this study, we found that kindlin-2, a widely distributed PTB domain protein, interacts with the C terminus of ␤ 3 CT at the TS 752 T and NITY 759 motifs and markedly enhances talin-induced integrin activation. Thus, kindlin-2 is identifi ed as a coactivator of integrins. Results and discussionTo address the functional signifi cance of the membrane-distal region of the ␤ 3 CT, we considered whether it might interact with intracellular regulator(s). A CHO cell line stably expressing ␣ IIb ␤ 3 was transfected with cDNAs encoding for wild-type or mutated ␤ 3 CT based on the rationale that these expressed constructs would compete for integrin binding partners. A similar strategy had been used previously to screen the ␤ CT binding partners essential for integrin activation ( Fenczik et al., 1997 ). In our studies, these ␤ 3 CT were expressed as chimeric constructs containing the extracellular domain of PSGL-1 so that expression levels of the various ␤ 3 CT could be verifi ed. As assessed by fl ow cytometry (FACS), PSGL-1 expression differed by less than 10%. The effects of the various ␤ 3 CT on ␣ IIb ␤ 3 -mediated cell spreading on immobilized fi brinogen were evaluated. Compared with cells expressing PSGL-1 alone, expression of the wild-type ␤ 3 CT chimera totally abolished ␣ IIb ␤...

PCAF histone acetylase plays a role in regulation of transcription, cell cycle progression, and differentiation. Here, we show that PCAF is found in a complex consisting of more than 20 distinct polypeptides. Strikingly, some polypeptides are identical to TBP-associated factors (TAFs), which are subunits of TFIID. Like TFIID, histone fold-containing factors are present within the PCAF complex. The histone H3- and H2B-like subunits within the PCAF complex are identical to those within TFIID, namely, hTAF(II)31 and hTAF(II)20/15, respectively. The PCAF complex has a novel histone H4-like subunit with similarity to hTAF(II)80 that interacts with the histone H3-like domain of hTAF(II)31. Moreover, the PCAF complex has a novel subunit with WD40 repeats having a similarity to hTAF(II)100.

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.