Identification and functional analysis of a new phosphorylation site (Y398) in the SH3 domain of Abi-1.

Abi-1 is an adaptor protein for Abelson kinase (c-Abl), and Abi-1 promotes the Abl-mediated phosphorylation of Mammalian Enabled (Mena) by binding both c-Abl and Mena. Here, we identified a new phosphorylation site (Y398) in the SH3 domain of Abi-1, and disruption of Y398, combined with the previously identified phosphorylation site Y213, significantly weakens the binding of Abi-1 to c-Abl. The SH3 domain of Abi-1 and the proline-rich domain of c-Abl are involved in this interaction. Abi-1 phosphorylation at both sites stimulates the phosphorylation of Mena through the activation of c-Abl kinase. The phosphorylation of Abi-1 also plays a role in enhancing the adhesion of Bcr-Abl-transformed leukemic cells.

Mass spectrometric imaging of ginsenosides localization in Panax ginseng root.

We performed mass spectrometric imaging (MSI) to localize ginsenosides (Rb(1), Rb(2) or Rc, and Rf) in cross-sections of the Panax ginseng root at a resolution of 100 microm using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Tandem mass spectrometry (MS/MS) of alkali metal-adducted ginsenoside ions revealed structural information of the corresponding saccharides and aglycone. MALDI-MSI confirmed that ginsenosides were located more in the cortex and the periderm than that in the medulla of a lateral root. In addition, it revealed that localization of ginsenosides in a root tip (diameter, 2.7 mm) is higher than that in the center of the root (diameter, 7.3 mm). A quantitative difference was detected between localizations of protopanaxadiol-type ginsenoside (Rb(1), Rb(2), or Rc) and protopanaxatriol-type ginsenoside (Rf) in the root. This imaging approach is a promising technique for rapid evaluation and identification of medicinal saponins in plant tissues.

Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase.

Reactive oxygen species (ROS) are implicated in plant innate immunity. NADPH oxidase (RBOH; for Respiratory Burst Oxidase Homolog) plays a central role in the oxidative burst, and EF-hand motifs in the N terminus of this protein suggest possible regulation by Ca(2+). However, regulatory mechanisms are largely unknown. We identified Ser-82 and Ser-97 in the N terminus of potato (Solanum tuberosum) St RBOHB as potential phosphorylation sites. An anti-phosphopeptide antibody (pSer82) indicated that Ser-82 was phosphorylated by pathogen signals in planta. We cloned two potato calcium-dependent protein kinases, St CDPK4 and St CDPK5, and mass spectrometry analyses showed that these CDPKs phosphorylated only Ser-82 and Ser-97 in the N terminus of St RBOHB in a calcium-dependent manner. Ectopic expression of the constitutively active mutant of St CDPK5, St CDPK5VK, provoked ROS production in Nicotiana benthamiana leaves. The CDPK-mediated ROS production was disrupted by knockdown of Nb RBOHB in N. benthamiana. The loss of function was complemented by heterologous expression of wild-type potato St RBOHB but not by a mutant (S82A/S97A). Furthermore, the heterologous expression of St CDPK5VK phosphorylated Ser-82 of St RBOHB in N. benthamiana. These results suggest that St CDPK5 induces the phosphorylation of St RBOHB and regulates the oxidative burst.

Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in brassica self-incompatibility.

In Brassica self-incompatibility, the recognition of self/nonself pollen grains, is controlled by the S-locus, which encodes three highly polymorphic proteins: S-locus receptor kinase (SRK), S-locus protein 11 (SP11; also designated S-locus Cys-rich protein), and S-locus glycoprotein (SLG). SP11, located in the pollen coat, determines pollen S-haplotype specificity, whereas SRK, located on the plasma membrane of stigmatic papilla cells, determines stigmatic S-haplotype specificity. SLG shares significant sequence similarity with the extracellular domain of SRK and is abundant in the stigmatic cell wall, but its function is controversial. We previously showed that SP11 binds directly to its cognate SRK with high affinity (K(d) = 0.7 nM) and induces its autophosphorylation. We also found that an SLG-like, 60-kD protein on the stigmatic membrane forms a high-affinity binding site for SP11. Here, we show that the 60-kD stigmatic membrane protein is a truncated form of SRK containing the extracellular domain, transmembrane domain, and part of the juxtamembrane domain. A transiently expressed, membrane-anchored form of SRK exhibits high-affinity binding to SP11, whereas the soluble SRK (eSRK) lacking the transmembrane domain exhibits no high-affinity binding, as is the case with SLG. The different binding affinities of the membrane-anchored SRK and soluble eSRK or SLG will be significant for the specific perception of SP11 by SRK.

Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases.

A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.