A facile, click chemistry-based approach to assembling fluorescent chemosensors for protein tyrosine kinases.

A group of fluorophore-labeled peptide substrates of Src kinases have been synthesized with the aid of click chemistry. Some of the generated peptides exhibit an increase in fluorescence upon phosphorylation and are capable of detecting Src kinases with high sensitivity and specificity. Their availability permits real-time activity measurement of aberrantly activated oncogenic Src kinases in the crude lysate of chronic myelogenous leukemia cells. These new chemosensor peptides are highly useful tools that can be used for high-throughput screening to search for small molecule inhibitors of Src kinases as potential therapeutics for cancer treatment.

Development of the procedures for high-yield expression and rapid purification of active recombinant Csk-homologous kinase (CHK): comparison of the catalytic activities of CHK and CSK.

Csk-homologous kinase (CHK) is an important endogenous inhibitor constraining the oncogenic actions of Src-family kinases (SFKs) in cells. It suppresses SFK activity by specifically phosphorylating the conserved regulatory tyrosine near the C-terminus of SFKs. In addition to phosphorylation, CHK employs a novel non-catalytic inhibitory mechanism to suppress SFK activity. This mechanism involves direct binding of CHK to the active forms of SFKs to form stable protein complexes. Since aberrant activation of SFKs contributes to cancer formation and progression, small-molecule inhibitors mimicking the non-catalytic inhibitory mechanism of CHK are potential anti-cancer therapeutics. Elucidation of the catalytic and regulatory properties and the structural basis of the CHK non-catalytic inhibitory mechanism would facilitate the development of these small-molecule inhibitors. To this end, we developed procedures for higher level expression in insect cells of active recombinant CHK with a hexa-histidine tag attached to its C-terminus (referred to as CHK-His(6)) and its rapid purification by a two-step method. Analyses by size-exclusion column chromatography and analytical ultracentrifugation revealed that the purified CHK-His(6) exists as a monomeric species in solution. Biochemical analyses demonstrated that CHK-His(6) exhibits efficiencies comparable to those of CSK in phosphorylating artificial protein and peptide substrates as well as an intact SFK protein. Our results indicate that the recombinant CHK-His(6) can be used for future studies to decipher the three-dimensional structure, and regulatory and catalytic properties of CHK.

Structural elements and allosteric mechanisms governing regulation and catalysis of CSK-family kinases and their inhibition of Src-family kinases.

C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) are endogenous inhibitors constraining the activity of the oncogenic Src-family kinases (SFKs) in cells. Both kinases suppress SFKs by selectively phosphorylating their consensus C-terminal regulatory tyrosine. In addition to phosphorylation, CHK can suppress SFKs by a unique non-catalytic inhibitory mechanism that involves tight binding of CHK to SFKs to form stable complexes. In this review, we discuss how allosteric regulators, phosphorylation, and inter-domain interactions interplay to govern the activity of CSK and CHK and their ability to inhibit SFKs. In particular, based upon the published results of structural and biochemical analysis of CSK and CHK, we attempt to chart the allosteric networks in CSK and CHK that govern their catalysis and ability to inhibit SFKs. We also discuss how the published three-dimensional structure of CSK complexed with an SFK member sheds light on the structural basis of substrate recognition by protein kinases.

Recombinant expression and characterization of five-domain Kazal-type serine proteinase inhibitor of black tiger shrimp (Penaeus monodon).

Four full-length complementary DNAs of Kazal-Type serine proteinase inhibitors (SPIs) were identified from hemocyte cDNA libraries of the black tiger shrimp Penaeus monodon and recognized as having 4 (SPIPm1) and 5 (SPIPm2, SPIPm3, and SPIPm4) Kazal domains. SPIPm2 encoding a complete 5-Kazal-domain inhibitor was expressed in the Escherichia coli system. Inhibitory activity of crude proteins against various serine proteases was tested using SPI activity gelatin with sodium dodecylsulfate polyacrylamide gel electrophoresis and inhibitory spectrum assays. A 32-kDa recombinant protein (rSPIPm2) showed inhibitory activity against trypsin, chymotrypsin, and subtilisin, but not elastase. Concordantly, inhibitory spectrum assays showed that crude rSPIPm2 strongly inhibited trypsin (89%) and chymotrypsin (70%), but less effectively inhibited subtilisin (8%), and did not inhibit elastase activity. Northern blot analysis of hemocyte total RNA showed 2 SPI transcripts of 1.6 and 1.7 kb in size. Tissue-specific expression using reverse transcriptase polymerase chain reaction suggests that SPIPm2 is exclusively expressed in hemocytes of P. monodon.