Effect of TGF- and Rock Inhibition on Phosphorylation of Myosin Light Chain - Lab Report Example

?Effect of TGF-? and Rock Inhibition on Phosphorylation of Myosin Light Chain Myosin light chains are distinct from heavy chains with their own properties. They are component of the macromolecular complexes of functional myosin enzymes. Phosphorylation of Myosin light chain (MLC) plays critical roles in several functions of cells such as smooth muscle contraction; motility and cellular morphogenesis (Chong et al., 2001).Determination of MLC phosphorylation is through balance between Rho-kinase activities and myosin phosphatase. An impaired balance that exists between myosin phosphatase and Rho-kinase activities induces the abnormal sustained MLC phosphorylation which contributes to particular vascular disease pathogenesis such as hypertension and vasospasm. However, the system dynamic principle underlying MLC phosphorylation regulation remains to be clarified. The main purpose of this experiment is to look at phosphorylation of myosin light chain and the way TGF-? and Rock inhibition affects phosphorylation in the MG63 cells using western blotting technique. Introduction Myosin consists of 6 polypeptide chains: two heavy chains that are identical and two light chains pairs. Myosin light chain 2 (MLC2), also referred to as myosin regulatory light chain (MRLC), RLC, has several isoforms that depends on its distribution. (Dan et al., 2002). In smooth muscle, there is phosphorylation of MLC2 by myosin light chain kinase at Ser19 and Thr18 in a Ca2+/calmodulin-dependent pathway. The phosphorylation is associated with the activity of myosin ATPase and contraction of smooth muscle. Smooth muscle MLC2 Ser19 is also phosphorylated by ROCK, it regulates stress fibers assembly. ROCK (Rho-kinase), an effector RhoA molecule, inhibits the activity of phosphatase and phosphorylates the myosin binding subunit (MBS) of myosin phosphatase. The inhibition increases myosin light chain (MLC) phosphorylation of myosin II, which seems to induce RhoA-mediated stress fibers assembly and focal adhesions. ROCK is as well known to phosphorylate directly MLC in vitro; smooth muscle MLC2 Phosphorylation at Ser9 and Ser1/Ser2 by cdc2 and PKC inhibits myosin ATPase activity. Phosphorylation by cdc2 controls the cytokinesis timing. The pathway of RhoA/Rho-kinase is a crucial component of TGF-beta-induced effects on MLC phosphorylation of endothelial cells (Harden et al., 1996). Both non-muscle and smooth muscle myosin II activity is controlled by phosphorylation with myosin regulatory light chain (MRLC, MLC20,MLC, Myl9). MLC Phosphorylation at Ser-19 and Thr-18 results to activation of myosin II motor activity and myosin filament stability increase. (Daniels et al., 1998). The activation has critical roles in several processes of cell motile. On the other hand, other sites of phosphorylation on MLC might inhibit the activity of myosin II. In MLC, PKC phosphorylates Thr-9 and Ser-1/Ser-2, the phosphorylation leads to decrease in the activated myosin II interaction with actin, and inhibits interaction of MLC with the myosin light-chain kinase activation site. Thus, myosin II activity inhibition through Ser-1/Ser-2 phosphorylation may have crucial roles in growth factor-induced actomyosin filaments reorganization. (Dechert et al., 2001). Materials and methods After the gel was transferred to the tank, it was filled with 1x running buffer:100ml 10x Trisglycine/SDS(10xrunning buffer)900ml dH2O.The samples were loaded,5ul Bio-Rad precision markers(kaleidoscope).The samples were boiled in 2x sample buffer at 95oc for 5minutes. Samples total volume per well were 30 ul, for protein, it was 40 ug of per well 7 ul for molecular weight marker Transfer of SDS gel onto the membrane First the blotting paper pieces were cut (7.5x9.5cm).Then the pieces of the membrane 7x9cm(PVDF membrane) were cut.The PVDF membrane was soaked in methanol for 10-20 seconds.A litre of transfer buffer was prepared:100ml 10xTris Glycine,700ml dH2O,200ml methanol The protein Separated was transferred onto PVDF membrane for 90 min at 100 V The gels were duplicated ( 3 transfer- 1 dye- 1 coomassie blue). This was done to enable transfer of good quality protein as well as to confirm the presence of protein of interest. Coomassie staining was performed between electrophases and western blotting. Coomassie blue contained 40% Methanol–10 % acetic acid - 50% water and its detection sensitivity was 50. Clear bands indicated good protein quality, de-staining was done for one hour with de-stainer that had same components without powder. Blocking of the membrane Three membranes were blocked; whereby two were stained and three transferred for three different antibodies. Blocking was done to block non proteins sites. Therefore when antibodies were added they bound only to proteins of interest. Blocking of the Membranes was done in 5% BSA/TBST buffer for 30 min. There was incubation of Primary rabbit anti-human antibody on the membrane overnight at 4C (1:1000 primary antibody dilutions in 5%BSA/TBST). Washing was later done 6 times x 5 min in TBST. There was then incubation of Secondary goat anti-rabbit HRP labeled antibody for thirty minutes (dilution of 1:5000 in 5 percent non-fat milk/TBST). 6 x 5 minute washing in TBST was done. Enhanced 20 mL chemiluminescence(ECL) substrate was applied to blots. Visualization of the Membranes was on UVP then images later taken. Results Before the transfer of the bands to the membrane the bands were visible on the gel, but after the transfer the bands were not visible on the gels. This means that the protein or bands were transferred successfully to the membrane as shown in figure 1. Figure 1: Shows the SDS gel and the membrane before and after transfer of the bands/proteins After the transfer of the protein bands to the membrane, the membrane was de-stained, blocked, incubated with Primary rabbit anti-human antibody then with Secondary goat anti-rabbit HRP labeled antibody. The visualization of the membranes was done on UVP after applying enhanced chemiluminescence (ECL) substrate to blots ,then later images were taken as shown in figure2 .The first and second membrane with Phosho-myosin light chain 2(Ser19), Phosho-myosin light chain 2(Thr 18/Ser 19) respectively had a lot of background binding of the antibodies as compared to the membrane with myosin light chain 2.The bands were visible in lane 1 ,3,4 of the myosin light chain but no bands seen in the other two membranes due to background binding of the antibodies. Figure 2: shows incubated membrane with antibodies visualized under UVP before optimization There was rerun of gels after optimization of the conditions as shown in figure 3,4 and 5.There was less background binding as compared to the previous run. Several bands were observed in every well. Figure 3: shows a membrane with Phospho MLC2(S19) after optimization. Figure 4: shows a membrane with Phospho MLC2(T18/S19) after optimization. Figure 5: Shows a membrane with Total MLC2 after optimization. Discussion Coomassie gels staining indicated that the protein preps quality was not very good. This is because the bands could not be visualized clearly as shown in figure 1 above. It’s expected that for a very good protein prep quality the bands should be visible. The staining solution prepared in the laboratory was not better than the commercially available stain. This is because the bands stained were not clearly distinct from the background. A commercially prepared stain is expected to bring out a very clear contrast between the background and the bands. Following western blot, to determine whether the proteins had been electrophoresed onto the PVDF membrane from the polyacrylamide gel was by observing the polyacrylamide gel if it had bands after the transfer process as well as the membrane. If the polyacrylamide gel has no bands following the transfer as for the case in this experiment as shown in figure 1, then it means that the proteins were transferred to the PVDF membrane. Similarly, if the membrane has visible bands following the transfer process, it is an indication that the proteins were transferred successfully. The membranes had a dark background or high background staining, these could have been caused by may be too concentrated sample, incorrect preparation/run of SDS-PAGE gel, Improper preparation of sample for gel loading. These problems can be minimized during optimization by following the right steps critically when preparing or running SDS-PAGE gel, proper sample preparation, use of optimal sample concentration it is known that Proper migration of protein on the gel is based on protein samples containing SDS and should be heated before loading it. Myosin light chain 2 protein molecular weight is approximately 18kDa.Therefore on the blot the band is seen between the 15kDa and 25kDa molecular weight of the ladder. Generally, there were several non-specifics binding to proteins by antibodies other than the MLC2 protein. This problem can be tackled by employing antigen affinity-purified antibodies or mono-specific antibodies, blocking in 5% milk, increase NaCl from 0.15 to 0.5M or milk from 2 to 5% in primary Antibody Solution concentrations. Can also try to increase on the number of washes, increase the concentration of Tween 20 in washing buffer from 0.1% to 0.5%. If the Film was overexposed, then the exposure time need to be reduced. If there was too strong target signal then need to wait for 5-10 minutes before re-exposing to the film. Basing on every treatment, there was little phosphorylation of MLC2 protein with control, no phosphorylation observed for Rock, phosphorylation of TGF-? was observed, Rock+TGF-? when combined, less phosphorylation was observed than TGF-? alone. The cells with phosphorylation of MLC2 protein were motile and active as compared to those which never had phosphorylation of MLC2 protein. Basing on the findings, it is clearly evident that the RhoA/Rho-kinase pathway is a crucial TGF-beta-induced effects component on endothelial MLC phosphorylation. Conclusion In conclusion, there was a lot of non specific binding on the membrane, the stain might have not been prepared so well because it was not able to clearly distinguish between the bands and the background, the transfer of proteins to the membrane was fairly good because the protein bands on the gel were not observed following the transferring process. Effective optimization was needed in order to obtain proteins of very good quality. There was little phosphorylation of MLC2 protein with control, no phosphorylation observed for Rock, phosphorylation of TGF-? was observed, Rock+TGF-? when combined, less phosphorylation was observed than TGF-? alone. Bibliography Chong C, Tan L, Lim L & Manser E (2001). The mechanism of PAK activation. Autophosphorylation events in both regulatory and kinase domains control activity. J Biol Chem 276, 17347–17353. Dan C, Nath N, Liberto M & Minden A (2002). PAK5, a new brain-specific kinase, promotes neurite outgrowth in N1E-115 cells. Mol Cell Biol 22, 567–577. Daniels RH, Hall PS & Bokoch GM (1998). Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells. EMBO J17, 754–764. Dechert MA, Holder JM & Gerthoffer WT (2001). p21-activated kinase 1 participates in tracheal smooth muscle cell migration by signaling to p38 Mapk. Am J Physiol 281, C123–132. Harden N, Lee J, Loh HY, Ong YM, Tan I, Leung T, Manser E & Lim L (1996). A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures. Mol Cell Biol 16, 1896–1908. Read More