It has been shown that ultrasound (US) stimulation accelerates fracture healing in animal models and in clinical studies. Here we found that US stimulation transiently increased the surface expression of ␣2, ␣5, ␤1, and ␤3 integrins in cultured osteoblasts, as shown by flow cytometric analysis and immunofluorescence staining. US stimulation increased prostaglandin E 2 formation and the protein and mRNA levels of cyclooxygenase-2 (COX-2). At the mechanistic level, anti-integrin ␣5␤1 and ␣v␤3 antibodies or rhodostomin, a snake venom disintegrin, attenuated the US-induced COX-2 expression. Phosphatidylinositol 3-kinase (PI3K) inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin also inhibited the potentiating action of US. US stimulation increased the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinases (ERK), p85 subunit of PI3K, and serine 473 of Akt. COX-2 promoter activity was enhanced by US stimulation in cells transfected with pCOX2-Luc. Cotransfection with dominant-negative mutant of FAK(Y397F), p85(⌬p85), Akt(K179A), or ERK2(K52R) inhibited the potentiating action of US on COX-2 promoter activity. Expression of mineralized nodule was lower in dominant-negative mutants of FAK, p85, and Akt-transfected clones than in vector-transfected control cells. Taken together, our results provide evidence that US stimulation increases COX-2 expression and promotes bone formation in osteoblasts via the integrin/ FAK/PI3K/Akt and ERK signaling pathway.

MST3 is a member of the sterile-20 protein kinase family with a unique preference for manganese ion as a cofactor in vitro; however, its biological function is largely unknown. Suppression of endogenous MST3 by small interference RNA enhanced cellular migration in MCF-7 cells with reduced expression of E-cadherin at the edge of migrating cells. The alteration of cellular migration and protruding can be rescued by RNA interference-resistant MST3. The expression of surface integrin and Golgi apparatus was not altered, but phosphorylation on tyrosine 118 and tyrosine 31 of paxillin was attenuated by MST3 small interfering RNA (siRNA). Threonine 178 was determined to be one of the two main autophosphorylation sites of MST3 in vitro. Mutant T178A MST3, containing alanine instead of threonine at codon 178, lost autophosphorylation and kinase activities. Overexpression of wild type MST3, but not the T178A mutant MST3, inhibited migration and spreading in Madin-Darby canine kidney cells. MST3 could phosphorylate the protein-tyrosine phosphatase (PTP)-PEST and inhibit the tyrosine phosphatase activity of PTP-PEST. We conclude that MST3 inhibits cell migration in a fashion dependent on autophosphorylation and may regulate paxillin phosphorylation through tyrosine phosphatase PTP-PEST.

No abstract

Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination of cis-and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn 356 might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10-and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions.

Interleukin enhancer binding factor (ILF) is a human transcription factor and a new member of the winged helix/forkhead family. ILF can bind to purine-rich regulatory motifs such as the human T-cell leukemia virus-long terminal region and the interleukin-2 promoter. Here we report the 2.4 Å crystal structure of two DNA binding domains of ILF (FOXK1a) binding to a 16-bp DNA duplex containing a promoter sequence. Electrophoretic mobility shift assay studies demonstrate that two ILF-DNA binding domain molecules cooperatively bind to DNA. In addition to the recognition helix recognizing the core sequences through the major groove, the structure shows that wing 1 interacts with the minor groove of DNA, and the H2-H3 loop region makes ionic bonds to the phosphate group, which permits the recognition of DNA. The structure also reveals that the presence of the C-terminal ␣-helix in place of a typical wing 2 in a member of this family alters the orientation of the C-terminal basic residues (RKRRPR) when binding to DNA outside the core sequence. These results provide a new insight into how the DNA binding specificities of winged helix/forkhead proteins may be regulated by their less conserved regions.The members of the extensive family of forkhead box (Fox) 3 proteins are transcription factors important for regulating cellular proliferation, transformation, differentiation, and longevity (1-3). They are expressed in various eukaryotic organisms with multiple domains that are specific for DNA binding, trans-activation, or trans-repression (4). The Fox proteins have a unique winged helix domain composed of ϳ100 evolutionarily well conserved amino acids essential for DNA recognition (5). Several three-dimensional structures of this DNA binding domain (DBD), including HNF-3␥ (FoxA3), Genesis (Foxd3), AFX (FOXO4), FREAC11 (FOXC2), and ILF-1 (FOXK1), have been determined using x-ray crystallography or NMR spectroscopy (6 -11). Most of these structures exhibit three ␣-helices, three ␤-strands, and two wing-like loops (12). The so-called winged helix DNA-binding motif of these proteins is named after the three-dimensional structure of the HNF-3␥-DNA complex in which two wing-like loops (wing 1 and wing 2) and helix 3 are involved in protein-DNA interactions (13).For many DNA-binding protein families, the differences in DNA binding specificity between family members are determined by the contact residues on recognition elements; the substitution of a DNA contact residue within family members leads to different binding specificity (14). However, transcription factors containing the winged helix-binding motif are exceptions (15). The DNA binding domains of Fox proteins exhibit a remarkable degree of sequence homology in their DNA binding region but a notable variability in their DNA recognition specificity (12). To date, more than 200 winged helix/forkhead proteins have been identified, but only two forkhead protein-DNA complexes have been reported (7, 13). Based on these two three-dimensional structures, it has been proposed that...