K-EGFP ( ) with antibodies against phosphotyrosine (pTyr). Proteins inside the lysates and immune complexes (IP) were probed with antibodies against PKAc or Syk. B, PKAc was immunoprecipitated from lysates of Syk-deficient MCF7-B ( ) or MCF7-B cells expressing Syk-EGFP ( ) or Syk-EGFP-NLS (NLS). Proteins inside the lysates and immune complexes (IP) have been probed with antibodies against PKAc, phosphotyrosine (pTyr), or Syk. A mock IP (m) was carried out within the absence of added antibody to detect possible nonspecific interactions. C, PKAc was immunoprecipitated from lysates of Syk-deficient MCF7-B ( ) or MCF7-A cells. Proteins in the lysates and immune complexes (IP) have been probed with antibodies against PKAc, phosphotyrosine (pTyr), or Syk. D, MCF7 cells stably expressing Syk-EGFP-NLS were viewed by fluorescence (left panel) or phase contrast microscopy (proper panel). WB, Western blotting.When released from its related regulatory subunits by cAMP, the catalytic subunit (PKAc) is active and capable of catalyzing the phosphorylation of various protein substrates. These substrates incorporate CREB, which PKAc phosphorylates after it translocates in to the nucleus (23). A fraction of Syk also is present within the nuclear compartment of B cells and breast epithelial cells (26, 28). To appear for phosphorylation of PKAc inside the nucleus, we generated an MCF7-B cell line that stably expressed Syk-EGFP-NLS, a kind of Syk-EGFP with a nuclear localization sequence derived from SV40 substantial T antigen appended to the C terminus (26). An examination of these cells by fluorescence microscopy confirmed a localization of SykEGFP-NLS mainly towards the nucleus (Fig. 6D). Western blot evaluation of anti-PKAc immune complexes isolated from cells expressing this nuclear localized Syk revealed a robust phosphorylation of PKAc on tyrosine (Fig. 6B).10876 JOURNAL OF BIOLOGICAL CHEMISTRYPhosphorylation of PKA by SykFIGURE 7. MS/MS spectrum coverage of PKAc peptide GPGDTSNFDDpYEEEEIR. The spectrum incorporates b-ions and y-ions. The spectrum ion coverage phosphoRS software program (Thermo) assigned 100 probability to Tyr-11 because the web site of phosphorylation.4-Bromo-6-(trifluoromethyl)-1H-indole Chemscene FIGURE 8.1445951-89-2 Chemscene CREB phosphorylation is inhibited by Syk in MCF7 cells. A, Sykdeficient MCF7-B cells (Syk ) or MCF7-B cells expressing Syk-EGFP or SykEGFP-NLS have been stimulated with forskolin for the indicated instances. The lysates were fractionated by SDS-PAGE and probed with antibodies against phoshoCREB (pCREB), CREB, Syk, GAPDH, and PKAc. B, relative levels of pCREB in lysates of forskolin-treated MCF7-B cells lacking Syk (Syk ) or expressing SykEGFP had been compared, as well as the ratios of the intensities of pCREB to CREB were plotted.PMID:23671446 The p values were calculated from Student’s t tests from 3 separate experiments. IB, immunoblot.FIGURE 9. CREB phosphorylation is enhanced by a Syk inhibitor. A, MCF7-B cells (Syk ) or MCF7-B cells expressing Syk-EGFP were pretreated with H-89 or piceatannol (PIC) and then stimulated with forskolin for the indicated times. The lysates have been fractionated by SDS-PAGE and probed with antibodies against phospho-CREB (pCREB), CREB, and Syk. B, lysates from Sykdeficient MCF7-B cells (Syk ) or MCF7-B cells expressing Syk-EGFP had been assayed for kinase activity against LRRASLG. The data represent the means and standard errors of 3 experiments.expressing Syk-EGFP to catalyze the phosphorylation of LRRASLG. As shown in Fig. 9B, PKA activity was lowered substantially in cells expressing Syk-EGFP as compared with Sykdeficient cells. Effe.