Characterization and purification of truncated human Rho-kinase II expressed in Sf-21 cells

Turner, Mary S.; Fen-Fen-Lin,; Trauger, John W.; Stephens, Jeffrey W.; LoGrasso, Philip V.

doi:10.1016/s0003-9861(02)00249-7

Cited by 31 publications

(22 citation statements)

References 39 publications

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“…This closed-to-open conformation of ROCK activation is similar to that of DMPK and MRCK activation [15,17], and is consistent with studies demonstrating that overexpression of various carboxyl-terminal constructs of ROCK or kinase-defective forms of full-length ROCK, function as dominantnegative ROCK mutants [4,5,21]. ROCKs can also be activated independently of Rho through amino-terminal transphosphorylation caused by protein oligomerization [15,22] or inhibited by other small GTP-binding proteins such as Gem and Rad [23].…”

supporting

confidence: 84%

ROCKs as therapeutic targets in cardiovascular diseases

Rikitake

Liao

2005

Expert Review of Cardiovascular Therapy

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There is growing evidence that Rho-kinases (ROCKs), the immediate downstream targets of the small guanosine triphosphate-binding protein Rho, may contribute to cardiovascular disease. ROCKs play a central role in diverse cellular functions such as smooth muscle contraction, stress fiber formation and cell migration and proliferation. Overactivity of ROCKs is observed in cerebral ischemia, coronary vasospasm, hypertension, vascular inflammation, arteriosclerosis and atherosclerosis. ROCKs, therefore, may be an important and still relatively unexplored therapeutic target in cardiovascular disease. Recent experimental and clinical studies using ROCK inhibitors such as Y-27632 and fasudil have revealed a critical role of ROCKs in embryonic development, inflammation and oncogenesis. This review will focus on the potential role of ROCKs in cellular functions and discuss the prospects of ROCK inhibitors as emerging therapy for cardiovascular diseases. Keywords contraction; endothelium; hypertension; inflammation; leukocyte; remodeling; Rho-kinase; smooth muscleThe small guanosine triphosphate (GTP)-binding proteins belonging to the Rho family regulate various aspects of cell shape, motility, proliferation and apoptosis [1]. Rho-kinases (ROCKs), which were one of the first downstream effectors of Rho to be discovered [2-4], were found to mediate RhoA-induced actin cytoskeletal changes through effects on myosin light-chain (MLC) phosphorylation [5,6]. ROCKs are protein serine/threonine kinases that share 45-50% homology with other actin cytoskeletal kinases such as myotonic dystrophy kinase (DMPK), myotonic dystrophy-related cdc42-binding kinase (MRCK) and citron kinase [1]. ROCKs consist of an amino-terminal kinase domain, followed by a mid-coiled-coil-forming region containing a Rho-binding domain (RBD) and a carboxy-terminal cysteine-rich domain (CRD) located within the pleckstrin homology (PH) motif (FIGURE 1). In mammalian systems, two ROCK isoforms have been identified. ROCK1, which is also known as ROKβ, and p160ROCK, which is located on chromosome 18 and encodes a 1354 amino acid protein [4,5]. ROCK2, which is also known as ROKα and sometimes confusingly called Rho-kinase, is located on chromosome 12 and contains 1388 amino acids [2,7,8]. ROCK1 and 2 share an overall 65% homology in amino acid sequence and 92% homology in their kinase domains

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supporting

confidence: 84%

ROCKs as therapeutic targets in cardiovascular diseases

Rikitake

Liao

2005

Expert Review of Cardiovascular Therapy

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“…However, PKC has isoforms and their roles in the Rho-kinase mediated Ca 2+ sensitization is not universal. The non-selective PKC inhibitor staurosporine is also a potent inhibitor of Rho-kinase with an IC 50 value 215-fold more potent than that of Y27632 (11). Thus, the strong inhibition of the CLM-, OXZ-, and ET-1-induced PP increases by staurosporine may further support the involvement of Rho-kinase in their action, although involvement of PKCs cannot be ruled out.…”

Section: +mentioning

confidence: 95%

Mechanisms Involved in the Contraction of Intrahepatic Portal Vein Branches by Clomipramine and Oxethazaine in Isolated Perfused Rat Livers

Masuda¹,

Edo²

2005

J Pharmacol Sci

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Abstract. Clomipramine (CLM) and oxethazaine (OXZ) were previously reported to increase portal pressure by contracting portal vein branches (PVBs) in isolated perfused rat liver. In the present study, to characterize the contractile mechanisms, the effects of Y27632, HA1077, staurosporine, papaverine, SKF96365, and sulindac sulfide on the portal pressure increase induced by CLM and OXZ were examined comparatively with those induced by endothelin-1. The results suggest that 1) intrahepatic PVBs employ a Rho-kinase-dependent pathway for sustained contraction, 2) CLM contracts PVBs by activating a Rho-kinase pathway and Ca 2+channels, and 3) OXZ acts primarily by promoting Ca 2+ entry through its ionophore-like action.Keywords: intrahepatic portal vein branch, Rho kinase, clomipramineIn previous studies (1, 2), we showed that the tricyclic antidepressant clomipramine (CLM) and the topical anesthetic oxethazaine (OXZ) increased portal pressure (PP) in isolated perfused rat liver (IPRL) independent of their pharmacological actions, and this PP increase was accompanied by marked intrahepatic flow redistribution as characterized by vital staining with fluorescent dyes, that is, peripheral mal-perfusion and short-circuited flow in the central portion of the liver. This flow redistribution was related to contraction of the intrahepatic portal vein branches (PVBs), but not to contraction of the hepatic vein. Endothelin-1 (ET-1) exhibited a similar intrahepatic flow disturbance in the comparative study (1, 2).Such intrahepatic flow redistribution, if induced in vivo by drugs, could lead to adverse effects due to a decrease in hepatic extraction and metabolism of drugs as well as hepatotoxicity due to hypoxia. Although drug toxicity based on such a mechanism has not been realized, it may be latently involved. Therefore, it is important to examine the mechanism of contraction of PVBs by drugs.IPRL is an indispensable tool to characterize contractile mechanisms of PVBs since the extrahepatic portal vein preparation, which is generally used in pharmacological studies, is much less sensitive to these drugs than IPRL (1, 2). At present, the exact localization of the contraction of PVBs by these drugs remains to be determined, although ET-1 is reported to cause localized constriction in the distal segments of preterminal portal venules (3). In the present study, to gain some insight into contractile mechanisms of intrahepatic PVBs by these drugs, we examined the effects of several smooth muscle relaxants and related compounds on the PP increase induced by CLM and OXZ in comparison to the effects on ET-1-induced PP increase.The method for preparation of IPRL and the perfusion system is the same as described in a previous paper (1). Male Sprague-Dawley rats (Japan SLC, Shizuoka) weighing 220 -240 g were used. The left and median lobes of the liver were excised and perfused with KrebsHenseleit bicarbonate buffer (containing 1.3 mM CaCl 2 ) saturated with 95% O 2 -5% CO 2 , in a non-recirculating constant flow (20 ml / min) system, a...

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“…The classical selective substrates for ROCK are myosin light chain, 19) and myosin light chain phosphatase, 20) which regulate actin contraction. Trauger's report 21) suggested that S6-derived peptide is also one of selective substrates for ROCK, and conformed by Turner et al 12) The constants of hROCK (Table 1) and IC 50 of Y-27632 were similar to Turner's report, 12) which suggested the FP assay system is effective and feasible to determine ROCK activity. Compared with hROCK-CD, the parameters of rROCK-CD (including activity, K m , K cat , and IC 50 of Y-27632) based on the activity assay system were also similar; which indicated that the purified rROCK-CD could be used for drug screening or further activity research.…”

Section: Discussionmentioning

confidence: 57%

“…Turner et al 12) have developed a method to harvest recombinant ROCK-CD of human (hROCK-CD) from Sf-21 cell, but the cost is relatively high and the procedure is time-consuming. Katoh et al 10) had tried to recombine rat ROCK-CD in E. coli in their experiment, but there are no further reports could verify whether this method is feasible.…”

Section: Discussionmentioning

confidence: 99%

“…Katoh et al 10) have conformed that 1-543 amino acid residua have catalytic function; further research suggested the kernel catalytic domain of ROCK (ROCK-CD) may be 92-354 amino acid residua. 11) Turner et al 12) reported a way to harvest recombinant human ROCK-CD from Sf-21 cell, which became a main purified ROCK source currently. However, this procedure is timeconsuming and at a relatively high cost; which makes its price relatively expensive.…”

mentioning

confidence: 99%

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Harvest Active Recombinant Rho Kinase from Escherichia coli

Duan

Wang

Chen

et al. 2006

Biological & Pharmaceutical Bulletin

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Rho kinase, known as Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK), 1) is one of the central regulatory molecules for cytoskeleton control and cell adhesion process.2) ROCK also involves in other cell functions such as apoptosis 3) and tumor invasiveness. 4) Rho kinase is thought to play an important role in a variety of cellar functions such as stress fiber formation, focal adhesion formation, cell aggregation, cell morphology, cytokinesis, cell migration, and Ca 2ϩ -sensitization in the smooth muscle. 2,5) Recent report showed that Rho kinase involves in regulation of the amyloid precursor protein processing.6) ROCK inhibitors are beneficial candidates for a wide range of diseases such as hypertension, inflammation, cancer, 4) erectile dysfunction, 7) and injury caused by ischemia and reperfusion. 8)There are two types of ROCK: ROCK-I (ROCK-b) mainly found in hepatocytes, and ROCK-II (ROCK-a) in central nerve system (CNS). ROCK is activated by binding with the activated form of a low molecular weight G protein, Rho. 9)Purified ROCK is important foundation for understanding the full function of ROCK, and is very useful in ROCK inhibitors screening. Katoh et al. 10) have conformed that 1-543 amino acid residua have catalytic function; further research suggested the kernel catalytic domain of ROCK (ROCK-CD) may be 92-354 amino acid residua.11) Turner et al. 12) reported a way to harvest recombinant human ROCK-CD from Sf-21 cell, which became a main purified ROCK source currently. However, this procedure is timeconsuming and at a relatively high cost; which makes its price relatively expensive. It is needed to improve a procedure to harvest the ROCK-CD at low price, at high quality, and at large quantity; harvesting active ROCK-CD from Escherichia coli (E. coli) may be the choice met the request. MATERIALS AND METHODS RT-PCRThe cDNA encoding the amino acids 5-552 of rat ROCK-II was cloned with polymerase chain reaction (PCR) techniques. Primers were designed according to rat ROCK-II sequence NM_009072 in GenBank.11) Briefly, Rat brain total RNA was extracted by TRIzol kit (Invitrogen, U.S.A.), cDNA was reverse transcribed by reverse transcriptase from rat brain total RNA. A partial cDNA fragment of rat ROCK-II, 1644 nt (13-1656) was amplified using Taq DNA Polymerase (Takara Biotech, Japan) with 10 pM of each primer (cycling conditions: 94°C for 60 s, 55°C for 60 s, 72°C for 90 s, 30 cycles, finally kept at 72°C for 20 min). Primer A was 5Ј-ATGAGCGGATCCCCGCCGACGGGGA-AAAT-3Ј and primer B was 5Ј-ACCTCTCTCGAGTATCT-GAGAGCTCTGGT-3Ј (the underline in primer A is BamHI site; and in primer B is XholI site). The PCR-amplified fragment was subcloned into pGEM-T Easy Vector (Promega, U.S.A.). Then, the cDNA of ROCK-CD was sequenced by the vector and confirmed as the encoding 5-552 amino acids.Positive Clone Screening ROCK-CD cDNA in pGEM-T Easy Vector was digested with BamHI and XhoI and ligated into pET28a(ϩ) expression vector (Novagen, U.S.A.). The pET28a(ϩ) contains a (His)6-tag at its N-t...

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Characterization and purification of truncated human Rho-kinase II expressed in Sf-21 cells

Cited by 31 publications

References 39 publications

ROCKs as therapeutic targets in cardiovascular diseases

ROCKs as therapeutic targets in cardiovascular diseases

Mechanisms Involved in the Contraction of Intrahepatic Portal Vein Branches by Clomipramine and Oxethazaine in Isolated Perfused Rat Livers

Harvest Active Recombinant Rho Kinase from Escherichia coli

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