Browsing by Subject "Enzyme Activation"

Now showing 1 - 3 of 3
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    SHP2 phosphatase promotes mast cell chemotaxis toward stem cell factor via enhancing activation of the Lyn/Vav/Rac signaling axis
    (The American Association of Immunologists, 2014-05-15) Sharma, Namit; Everingham, Stephanie; Ramdas, Baskar; Kapur, Reuben; Craig, Andrew W.B.; Department of Pediatrics, IU School of Medicine
    SHP2 protein-tyrosine phosphatase (encoded by Ptpn11) positively regulates KIT (CD117) signaling in mast cells and is required for mast cell survival and homeostasis in mice. In this study, we uncover a role of SHP2 in promoting chemotaxis of mast cells toward stem cell factor (SCF), the ligand for KIT receptor. Using an inducible SHP2 knockout (KO) bone marrow-derived mast cell (BMMC) model, we observed defects in SCF-induced cell spreading, polarization, and chemotaxis. To address the mechanisms involved, we tested whether SHP2 promotes activation of Lyn kinase that was previously shown to promote mast cell chemotaxis. In SHP2 KO BMMCs, SCF-induced phosphorylation of the inhibitory C-terminal residue (pY507) was elevated compared with control cells, and phosphorylation of activation loop (pY396) was diminished. Because Lyn also was detected by substrate trapping assays, these results are consistent with SHP2 activating Lyn directly by dephosphorylation of pY507. Further analyses revealed a SHP2- and Lyn-dependent pathway leading to phosphorylation of Vav1, Rac activation, and F-actin polymerization in SCF-treated BMMCs. Treatment of BMMCs with a SHP2 inhibitor also led to impaired chemotaxis, consistent with SHP2 promoting SCF-induced chemotaxis of mast cells via a phosphatase-dependent mechanism. Thus, SHP2 inhibitors may be useful to limit SCF/KIT-induced mast cell recruitment to inflamed tissues or the tumor microenvironment.
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    Unraveling the Complexities of DNA-Dependent Protein Kinase Autophosphorylation
    (American Society for Microbiology (ASM), 2014-06) Neal, Jessica A.; Sugiman-Marangos, Seiji; VanderVere-Carozza, Pamela; Wagner, Mike; Turchi, John; Lees-Miller, Susan P.; Junop, Murray S.; Meek, Katheryn; Department of Medicine, IU School of Medicine
    DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a “hinge” region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ.