p94, a muscle-specific member of calpain family, is unique in that it undergoes rapid and exhaustive autolysis with a half-life of less than 1 h resulting in its disappearance from muscle. Recently, p94 was shown to be responsible for limb girdle muscular dystrophy type 2A. To elucidate the muscular proteolytic system mediated by p94 and to solve the mystery of its unusually rapid autolysis, we searched for p94-binding proteins by the two-hybrid system. Although calpain small subunit plays a crucial role for regulation of ubiquitous calpains, it did not associate with p94. After a screening of skeletal muscle library, connectin (or titin), a gigantic filamentous protein spanning the M-to Z-lines of muscle sarcomere, was found to bind to p94 through a p94-specific region, IS2. The connectin-insoluble fraction of washed myofibrils contained full-length intact p94, suggesting that connectin regulates p94 activity.Proteolysis in cytosol is a key reaction to modulate various intracellular protein functions such as signal transduction, protein turnover, and cell structure. Calpain, Ca 2ϩ -dependent cysteine proteinase (EC 3.4.22.17), is one of the major intracellular proteinases known as interacting various protein kinases, transcription factors, and cytoskeletal proteins (1-5). Ubiquitous -and m-calpains, dimers of a large catalytic (CL 1 (6) and mCL (7), respectively) and small regulatory (30K (8)) subunit. Recently, we found that the ubiquitous calpain large subunit monomer can express full proteolytic activity, and that 30K dissociates from the large subunit upon activation by Ca 2ϩ (9, 10). In other words, 30K together with calpastatin, a specific proteinaceous inhibitor for calpain, play pivotal roles in regulation of calpain activity.p94 is a muscle-specific member of the calpain large subunit family, and distinct not only from the other members but also from other proteases in that it autolyzes very rapidly and extensively leading to almost complete disappearance right after translation even in the presence of EGTA and leupeptin as observed in vitro (11,12). Ubiquitous calpains as well as many other proteases also undergo autolysis at the NH 2 terminus, but only to a limited extent. Quite recently, p94 was identified as responsible for limb girdle muscular dystrophy type 2A (LGMD2A), the first demonstration of the involvement of an enzyme in muscle dystrophy (13). To elucidate physiological meaning of this exhaustive autolysis and the molecular mechanism connecting LGMD2A and p94 function, it is important to clarify the substrates of p94 and the manner to regulate the proteolytic activity of p94.30K is the first candidate to bind to and regulate p94, since the calmodulin-like Ca 2ϩ -binding domains of CL and mCL (see Fig. 2), which are the binding sites for 30K, are highly homologous to p94 (5). Analysis of p94 at the protein level, however, is very difficult because of the extremely rapid autolysis, and, thus, we examined using the yeast two-hybrid system (14). As a result, 30K was revealed not to bind to p94...

Integrins are cell-surface receptors that mediate and coordinate cellular responses to the extracellular matrix (ECM). Cellular signalling pathways can regulate cell adhesion by altering the affinity and avidity of integrins for ECM. The Ras family of small G proteins, which includes H-ras, R-ras and Rap, are important elements in cellular signalling pathways that control integrin function.

p94, a skeletal muscle-specific calpain, has attracted much attention because its gene is responsible for limb-girdle muscular dystrophy type 2A. p94, however, has not been characterized at the protein and enzyme levels, owing to its very rapid autolysis. In the present study, a purification procedure for p94 was first established by using a recombinant inactive p94 expressed in COS cells in which the active site cysteine residue was changed to serine [p94(C129S)]. The isolation of native p94 from rabbit skeletal muscle by the established method with conventional procedures was extremely difficult because p94 became highly unstable in a crude extract on the addition of NaCl for separation. Purification of native p94 was possible with an antibody-affinity column but only as an inactive enzyme; p94(C129S) was purified as a homodimer. Characterization of p94, especially autolysis, was performed with partly purified native p94 and p94(C129S). The autolysis of p94, which consisted at least partly of an intermolecular reaction, proceeded in three consecutive steps; 60 and 58 kDa fragments were produced as intermediates before a stable 55 kDa fragment appeared. Autolysis of p94 was regarded as a degradative step rather than for the activation of the enzyme. All the autolysis cleavage sites were located in the p94-specific insertion sequence 1 region, which explains why p94 is unstable compared with the other calpains. The autolysis sites in p94 clearly showed a different specificity relative to the autolytic and proteolytic cleavage sites of the ubiquitous mu- and m-calpains, in its preference for residues at the P3 to P1' sites, indicating a distinct substrate specificity and function for the muscle enzyme.

No abstract

Abstract-Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to atheroprotection. Here we report that localized ␣4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress-induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial ␣4 integrins became phosphorylated on Ser 988 . In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced ␣4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a nonphosphorylatable ␣4(S 988 A) mutation failed to align in response to shear stress, thus establishing ␣4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent ␣4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress. (Circ Res. 2008;103:177-185.)