In humans, ROCK1 and ROCK2 both contain 33 exons and are located on chromosome 18 (18q11.1) and 2 (2p24) respectively. The ROCK1 open reading frame encodes 1354 amino acids, whereas ROCK2 encodes 1388 amino acids. ROCK2 also has a reported splice variant, preferentially expressed in skeletal muscle, which results in the inclusion of 57 additional amino acids. 7 The two ROCK homologs shares 64% identity in their primary amino acid sequences, with the highest homology (92%) within the kinase domains and the coiled-coil domains being the most diverse (55%). 5 The kinase domains of ROCK are closely related to many homologous domains in this family, including dystrophia myotonica protein kinase (DMPK), myotonic dystrophy kinase related-Cdc42 related kinases (MRCK) α and β, and citron Rho-interacting kinase (CRIK) . To date, the crystal structures of the kinase domains from ROCK1, 8 ROCK2, 9 MRCKβ, 10 and DMPK 11 have been determined, which has highlighted the high degree of tertiary as well as primary similarity. N-terminal and carboxyl-terminal extensions of the ROCK kinase domains are essential for catalytic activity. 4,8,9 The ROCK kinase domains are located in the N-terminal region, which is followed by a central ~600 amino acid long amphipathic α-helix forming a coiledcoil region (Fig. 1).12 At the carboxyl-terminal region, there is a split pleckstrin homology (PH) domain, which is bisected by an internal cysteine-rich zinc finger-like motif domain (CRD). The two separate PH portions assemble together to form a typical PH domain that is attached by two short linkers to a separate CRD. 13The canonical Rho binding domain (RBD) forms a parallel coiled coil dimer, as revealed by crystal structure determinations, and binds exclusively to the switch I and switch II regions of GTP-bound active RhoA and RhoC.14,15 Two additional Rhointeracting domains were identified that can tightly interact with RhoA, which may cooperatively contribute to binding. 16 Crystal structure studies revealed that ROCK has two dimerization domains: the ~70 residue N-terminal dimerization region 8,9 and the coiled-coil helical regions.12 Charged residues in the coiled-coil might function as a hinge that allows the N-terminal kinase domains to interact with C-terminal inhibitory regions. Structural determination of full-length ROCK protein crystals will ultimately reveal how the various domains interact and the mechanism of auto-inhibition. Regulation of ROCK ActivityAlthough ROCK1 and ROCK2 have highly related functional domains and significant amino acid identity, they are regulated both by common means as well as mechanisms unique to ROCK1 or ROCK2.

Apoptosis is a fundamental homeostatic mechanism essential for the normal growth, development and maintenance of every tissue and organ. Dying cells have been defined as apoptotic by distinguishing features, including cell contraction, nuclear fragmentation, blebbing, apoptotic body formation and maintenance of intact cellular membranes to prevent massive protein release and consequent inflammation. We now show that during early apoptosis limited membrane permeabilization occurs in blebs and apoptotic bodies, which allows release of proteins that may affect the proximal microenvironment before the catastrophic loss of membrane integrity during secondary necrosis. Blebbing, apoptotic body formation and protein release during early apoptosis are dependent on ROCK and myosin ATPase activity to drive actomyosin contraction. We identified 231 proteins released from actomyosin contraction-dependent blebs and apoptotic bodies by adapted SILAC (stable isotope labeling with amino acids in cell culture) combined with mass spectrometry analysis. The most enriched proteins released were the nucleosomal histones, which have previously been identified as damage-associated molecular pattern proteins (DAMPs) that can initiate sterile inflammatory responses. These results indicate that limited membrane permeabilization occurs in blebs and apoptotic bodies before secondary necrosis, leading to acute and localized release of immunomodulatory proteins during the early phase of active apoptotic membrane blebbing. Therefore, the shift from apoptosis to secondary necrosis is more graded than a simple binary switch, with the membrane permeabilization of apoptotic bodies and consequent limited release of DAMPs contributing to the transition between these states.

Apoptotic cell clearance facilitates the removal of aged, damaged, infected or dangerous cells although minimizing perturbation of surrounding tissues, and is a vital process in the development and homeostasis of multicellular organisms. Importantly, failure to correctly execute programmed cell death and subsequent corpse clearance is broadly associated with chronic inflammatory and/or autoimmune diseases such as systemic lupus erythematosus. Apoptotic cells develop dramatic morphological changes including contraction, membrane blebbing and apoptotic body formation, which were among the first and most readily identifiable features of cellular suicide. However, understanding the purpose of apoptotic cell morphological changes has proven to be elusive, and recent studies have made somewhat surprising, and occasionally opposing, conclusions about the contribution of blebbing to phagocytic clearance and prevention of inflammatory/autoimmune disease. We review the evidence indicating how apoptotic blebs actively promote corpse recognition, uptake, and generation of auto-reactive antibodies.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a Kras G12D/p53 R172H mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three‐dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK‐induced genes that facilitate extracellular matrix remodeling, with greatest fold‐changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13. MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three‐dimensional contexts. Treatment of Kras G12D/p53 R172H PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor‐associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth.

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating Aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour suppressive mechanism.

Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3β or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.

Continuous illumination (CI) induces an oxidative stress of the retina which is involved in light-induced retinal degeneration (LIRD). As the increase of glucocorticoids (GC) could also collaborate in the damage, adrenalectomized (ADX) and sham-operated rats (control, CTL) were submitted to CI, and their eyes were studied at light and electron microscopic levels. After CI, ADX retinas were significantly thicker than CTL retinas. Retinal alterations appeared earlier and were severer in CTL than in ADX retinas. Corticosterone levels increased gradually in the sera of CTL rats along CI. These results suggest that adrenalectomy attenuates LIRD, supporting the hypothesis.

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