A force-activated kinase in a catch smooth muscle

Abstract

Permeabilized anterior byssus retractor muscles (ABRM) from Mytilus edulis were used as a simple system to test whether there is a stretch dependent activation of a kinase as has been postulated for titin and the mini-titin twitchin. The ABRM is a smooth muscle that shows catch, a condition of high force maintenance and resistance to stretch following stimulation when the intracellular Ca++ concentration has diminished to sub-maximum levels. In the catch state twitchin is unphosphorylated, and the muscle maintains force without myosin crossbridge cycling through what is likely a twitchin mediated tether between thick and thin filaments. In catch, a small change in length results in a large change in force. The phosphorylation state of an added peptide, a good substrate for molluscan twitchin kinase, with the sequence KKRAARATSNVFA was used as a measure of kinase activation. We find that there is about a two-fold increase in phosphorylation of the added peptide with a 10% stretch of the ABRM in catch. The increased phosphorylation is due to activation of a kinase rather than to an inhibition of a phosphatase. The extent of phosphorylation of the peptide is decreased when twitchin is phosphorylated and catch force is not present. However, there is also a large increase in peptide phosphorylation when the muscle is activated in pCa 5, and the catch state does not exist. The force-sensitive kinase activity is decreased by ML-9 and ML-7 which are inhibitors of twitchin kinase, but not by the Rho kinase inhibitor Y-27632. There is no detectable phosphorylation of myosin light chains, but the phosphorylation of twitchin increases by a small, but significant extent with stretch. It is possible that twitchin senses force output resulting in a force-sensitive twitchin kinase activity that results in autophosphorylation of twitchin on site(s) other than those responsible for relaxation of catch.

Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase

“…Calmodulin has been shown to bind the CRD of TK and TwcK in vitro (10), but it does not lead to significant activation (9,11). Recently, in agreement with the involvement of these kinases in stretch-mediated pathways, it has been suggested that the tension that builds up in the cytoskeleton during muscle function pulls the CRD from the kinase, causing its release by mechanical unfolding (3,4). The stretch-activation of human TK studied by atomic force microscopy (AFM) and molecular dynamics (MD) simulations (12)(13)(14)(15) indicated that strain might lead to an ordered sequence of conformational changes that unwind the CRD, leaving an active kinase core.…”

“…Further, the conserved Ig-Ig-Fn tandem preceding TK is a receptor site for the ubiquitin ligase MuRF1 (5, 6), which is linked to the atrophy that results from muscle disuse. In TwcK, mechanical stress has been correlated with increased kinase activity (4). These data suggest that titin-like kinases form mechanosensory signaling nodes central to myofibril regulation.…”

“…This conservation suggests that titin-like kinases are important members of signaling networks in the sarcomere, although little is known about their cellular context. Titin kinase (TK) and twitchin kinase (TwcK) are the best studied members of the family and have been linked to stretch-activated pathways in muscle (3,4). In TK, the strain that develops in the cytoskeleton during muscle activity has been proposed to induce a conformational state that is recognized by the autophagosome receptors Nbr1 and p62 and the ubiquitin ligase MuRF2, which thereby are recruited to titin.…”

Catch Muscle Myorod Modulates ATPase Activity of Myosin in a Phosphorylation-Dependent Way

“…The phosphorylation of myorod at the two different sites of the molecule indicates the possibility of regulation of its unknown function. For example, twitchin is seems to be a force-sensitive kinase, which is thought to be activated by stretch that results in its autophosphorylation [ 26 ]. The idea that twitchin is a mechanical link interconnecting thin and thick filaments [ 12 ] though its interaction with actin [ 18 ] and myosin [ 12 ; 27 ] or myorod and paramyosin [ 12 ] supports this possibility.…”

Mechanism and Function of the Catch State in Molluscan Smooth Muscle: A Historical Perspective

“…It is now clear that the 5-HT-induced relaxation of the catch state is mediated by cyclic AMP(cAMP), which activates protein kinase A to result in the phosphorylation of a high molecular mass (~600 kDa) protein, twitchin [ 20 , 41 , 42 ]. Butler et al [ 43 ] recently reported that, in skinned ABRM fibers, twitchin phosphorylation increases in stretched fibers, suggesting that twitchin senses tension in the ABRM, to result in an increase in twitchin kinase activation. However, this phenomenon is related to the relaxation of the catch state, but not to the establishment of the catch state.…”