| Summary: | Myocyte stress 1 (MS1) also known as Striated Muscle Activator of Rho Signalling (STARS) and Actin-binding Rho activating protein is a stress responsive, muscle specific protein expressed in cardiac, skeletal and smooth muscle. MS1 was first observed to increase in mRNA levels during early states of pressure overload induced left ventricular hypertrophy, making MS1 sensitive to extracellular stress. MS1 is highly involved in actin dynamics, where polymerization of actin leads to the regulation of the myocardin related transcription factor-A and the serum response factor (MRTF-SRF). The SRF pathway plays a critical role in the regulation of the skeletal muscle. While in the heart, MS1 is thought to be implicated in hypertrophic signalling and cardiac remodelling. Previous studies in the lab have shown that MS1 increased in mRNA levels during simulated ischaemia/reperfusion injury but levels were attenuated with the addition of JNK inhibitor SP600125 during simulated ischaemia/reperfusion injury. Although we know MS1 is involved in actin dynamics due to its ability to bind actin at the C-terminal as a result of actin binding domains (located between 234-375 a.a) and also bind actin binding proteins ABLIM-2 and 3, there is limited information on how MS1 becomes upregulated and its specific function. In this study we wanted to investigate the effect of various stimuli on MS1 promoter activation with the use of luciferase reporter assays. The MS1 promoter was responsive to sorbitol induced osmotic stress, oxidative stress by serum deprivation and hypertrophic agonist phenylephrine. All of these are well known activators of the stress activated protein kinases (SAPKs); JNK and p38. There may be a link between MAPK activation and MS1 regulation. Investigation of other interacting partners of MS1 was proposed to give some insight into the function of MS1. Binding assays using purified MS1 fragments were used to look at potential interactions in the heart. Interestingly, novel myofibrillar proteins were pulled out of heart extract and identified by mass spectrometry as Myosin-6, troponin I, troponin T, α-tropomyosin, myosin LC2 and actin. We observed potential phosphorylation sites, located within the N-terminus of MS1. In vitro kinase assays using activated JNK, p38 and ERK, allowed for phosphorylation of MS1. Three novel phosphorylation sites Thr24, Thr62 and Ser77 were identified by mass spectrometry. Immunofluorescence studies were used to determine whether phosphorylation alters MS1 subcelluar distribution or interaction with actin. Co-localisation was observed between MS1 and HA-JNK at the cell membrane where there was evidence of membrane ruffling and actin stress fibres present at the periphery. All of these findings in this study are novel and imply that MS1 may be involved in the MAPK pathway and also play critical roles in contractile function, muscle development and cell motility, where phosphorylation may be responsible for its ability to interact with myofibrillar proteins.
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