Acyltransferases

J Biol Chem 277: 22115C22118, 2002

J Biol Chem 277: 22115C22118, 2002. intriguingly this response was fully abolished in the AMPK KD mice. Thus, TBC1D1 is differentially regulated in response to insulin and contraction. This study provides genetic evidence to support an important role for AMPK in regulating TBC1D1 in response to both of these physiological stimuli. at 4C for Docetaxel (Taxotere) 20 min. Supernatants were collected and snap-frozen in liquid nitrogen and stored at ?80C for later Docetaxel (Taxotere) analysis. Total protein concentrations were analyzed by the bicinchoninic acid method (Pierce Biotechnology, Rockford, IL). Immunoprecipitation. Total TBC1D1 protein was immunoprecipitated (IP) from 300 g of total muscle lysate protein using 1 g of TBC1D1 antibody, as previously described (4). Protein G-agarose beads (Sigma Aldrich, St. Louis, MO) were washed three times in PBS and added to the muscle lysate. Mixtures of lysate, antibody and protein G beads were rotated end over end overnight at 4C, and beads were subsequently washed twice in ice-cold PBS and placed in SDS sample buffer for 5 min at 96C. SDS-PAGE and western blot analyses. Muscle lysate proteins were separated by SDS-PAGE and transferred (semidry) to PVDF membranes (Immobilon Transfer Membranes; Millipore, Bagsvaerd, Denmark). Membranes were then blocked for 1 Docetaxel (Taxotere) h at room temperature (RT) in TBST + 1% BSA (wt/vol, pH 7.4) for TBC1D1 phosphospecific antibodies and in TBST + 2% skim milk powder (wt/vol, pH 7.4) for all other proteins. Blocked membranes were probed with primary antibodies (see 0.05 Keratin 18 (phospho-Ser33) antibody was considered statistically significant. Open in a separate window Fig. 1. TBC1D1 protein expression in muscle from wild-type (WT) and AMPK kinase dead (KD) mice. = 8). = 16). Data are expressed relative to WT soleus and are means SE. **Significant difference between soleus and EDL muscle, 0.01; ??significant difference between WT and AMPK KD mice, 0.01. and show that the anti-PAS antibody predominantly recognizes TBC1D1 compared with TBC1D4 in mouse EDL muscle and predominantly TBC1D4 compared with TBC1D1 in mouse soleus muscle. Table 1. Effects of contraction and insulin on AMPK phosphorylation, activity, and expression and ACC phosphorylation = 7C8. AMPK Thr172 and acetyl-CoA carboxylase (ACC) Ser227 phosphorylation (p) and AMPK1 and -2 protein expression in EDL muscle from WT and AMPK kinase dead (KD) mice in response to contraction or insulin. AMPK1 and -2 activity in muscle from WT and AMPK KD mice in response to contraction. */**Difference between basal and stimulated muscle, 0.05/0.01; ?/??difference between WT and AMPK KD mice, 0.05/0.01; (?)differences between WT and AMPK KD mice are borderline significant (= 0.06). Table 2. Effects of contraction and insulin on Akt phosphorylation and expression = 8. Akt phosphorylation on Thr308 and Ser473 and Akt protein expression in EDL muscles from WT and AMPK KD mice in response to contraction or insulin. **Difference between basal and insulin-stimulated muscle, 0.01. Table 3. Effects of AICAR, contraction, and insulin on AMPK, ACC, and Akt phosphorylation in WT EDL muscle = 8. Thr308 and Ser473: increase in phosphorylation was higher in insulin-stimulated muscle than AICAR- or contraction-stimulated muscle (? 0.05). Combining insulin and AICAR or contraction did not increase phosphorylation above insulin levels. pAMPK Thr172: contraction increased phosphorylation to a higher extent than AICAR ( 0.05); combining AICAR and contraction increased phosphorylation above AICAR (? 0.05) but not contraction levels. pACC Ser227: AICAR and contraction combined increased phosphorylation to a higher extent than AICAR or contraction alone (a 0.05). */** 0.05/0.01 toward Docetaxel (Taxotere) basal. Table 4. Effects of wortmannin on insulin- and contraction-stimulated Akt Thr308, Akt Ser473, and AMPK Thr172 phosphorylation in WT EDL muscle = 8. Akt phosphorylation on Thr308 and Ser473 and AMPK phosphorylation on Thr172 in EDL muscles from WT mice in response to contraction or insulin in the presence or absence of wortmannin. */**Difference between basal and contraction/insulin-stimulated muscle, 0.05/0.01. ??Difference between control and wortmannin-incubated muscle, 0.01. RESULTS Protein expression of TBC1D1. A five- to sixfold higher TBC1D1 protein expression in EDL muscle compared with soleus (Fig. 1) led us.