PTB Domain-Directed Substrate Targeting in a Tyrosine Kinase from the Unicellular Choanoflagellate Monosiga brevicollis

Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition

Activation of the nonreceptor protein tyrosine kinase Ack by multiple extracellular stimuli

Ack/Ack1 is a nonreceptor protein tyrosine kinase that comprises a tyrosine kinase core, an SH3 domain, a Cdc42-binding region, a Ralt homology region, and a proline-rich region. Here we describe a detailed characterization of the Ack protein as well as the chromosomal localization of human Ack (chromosome 3q29) and the primary structure of murine Ack. We demonstrate that Ack is ubiquitously expressed, with highest expression seen in thymus, spleen, and brain. Activation of integrins by cell adhesion on fibronectin leads to strong tyrosine phosphorylation and activation of Ack. Upon cell stimulation with EGF or PDGF, Ack is tyrosine-phosphorylated and recruited to activated EGF or PDGF receptors, respectively. A pool of endogenous Ack molecules is constitutively tyrosine-phosphorylated, even in starved cells. Moreover, tyrosine-phosphorylated Ack forms a stable complex with the adapter protein Nck via its SH2 domain. Finally, we have characterized a membrane-targeting sterile α motif-like domain in the amino terminus of Ack. Using several Ack mutants, we show that the amino-terminal and CRIB domains are necessary for Ack autophosphorylation, whereas the SH3 domain appears to have an autoinhibitory role. These experiments suggest a functional role for Ack as an early transducer of multiple extracellular stimuli

Summary of dengue diagnostic results obtained from patients and their corresponding/accompanying household members.

<p>Summary of dengue diagnostic results obtained from patients and their corresponding/accompanying household members.</p

Supervised hierarchical correlation clustering of 27 sample pairs.

<p>The heat map shows the gene expression of samples in columns, with a dendogram representing their similarity based on correlation. The samples' mean-subtracted log2 signal values were clustered using complete linkage (maximum) method and the genes' expression is shown with blue representing down-regulation, white for no change (zero), and red for up-regulation.</p

Asymptomatic versus patient paired volcano plot to determine differentially expressed genes.

<p>The <i>x</i>-axis shows the paired-sample t-test log₂ fold change of asymptomatic versus symptomatic patients, whereas the <i>y</i>-axis shows the −log₁₀ of the paired t-test p-values of asymptomatic versus symptomatic patients. Each dot represents a gene and the dot color depicts the gene's expression as shown by the standard deviation (SD) of the paired t-test log₂ fold change, e.g. shades of blue for less than or equal to −1 SD, grey for between −1 SD and +1 SD, and red for greater than or equal to +1 SD. A total of 345 genes were differentially expressed at more than 2 and less than −2 linear fold changes (p<0.01) and were considered statistically significant.</p

Summary of plaque reduction neutralization test (PRNT) results obtained from patients and their corresponding/accompanying household members.

<p>The numbers within parentheses indicate percentage.</p

53 of the selected canonical pathways studied, with genes up- and down-regulated in the subclinical/asymptomatic dengue are indicated.

<p>The pathways have been grouped according to the broad categories of reported dengue host defence mechanisms. The gene symbols are based on HUGO Gene Nomenclature Committee (HGNC) symbols. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092240#pone.0092240.s006" target="_blank">Table S1</a> for more details as it also includes the list of genes that showed no change for each of the pathways listed and provides linear fold change for genes discussed in the manuscript.</p

The PX-BAR membrane-remodeling unit of sorting nexin 9

Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism