Ser1333 phosphorylation indicates ROCKI activation
© Chuang et al.; licensee BioMed Central Ltd. 2013
Received: 2 July 2013
Accepted: 22 October 2013
Published: 29 October 2013
Two isoforms of Rho-associated protein kinase (ROCK), ROCKI and ROCKII, play a pivotal role in regulation of cytoskeleton and are involved in multiple cellular processes in mammalian cells. Knockout mice experiments have indicated that the functions of ROCKI and II are probably non-redundant in physiology. However, it is difficult to differentiate the activation status of ROCKI and ROCKII in biological samples. Previously, we have identified phosphorylation site of ROCKII at Ser1366 residue sensitive to ROCK inhibition. We further investigated the activity-dependent phosphorylation site in ROCKI to establish the reagents that can be used to detect their individual activation.
The phosphorylation site of ROCKI sensitive to its inhibition was identified to be the Ser1333 residue. The ROCKI pSer1333-specific antibody does not cross-react with phosphorylated ROCKII. The extent of S1333 phosphorylation of ROCKI correlates with myosin II light chain phosphorylation in cells in response to RhoA stimulation.
Active ROCKI is phosphorylated at Ser1333 site. Antibodies that recognize phospho-Ser1333 of ROCKI and phospho-S1366 residues of ROCKII offer a means to discriminate their individual active status in cells and tissues.
KeywordsRho-associated protein kinase (ROCK) RhoA Marker
Two isoforms of Rho-associated protein kinase (ROCK), ROCKI (also called ROKβ) and ROCKII (also known as Rho kinase and ROKα) have been identified as RhoA-GTP interacting proteins in mammals [1, 2]. They are serine/threonine kinases important for regulation of actin dynamics and cytoskeleton organization [3–5]. These two human kinases share 64% homology in amino acid sequence with 89% identity in the catalytic kinase domain . They contain a Rho-binding domain (RBD) in the coiled-coil region and a pleckstrin homology (PH) domain in the C-terminal region, which folds back onto the N-terminal kinase domain to autoinhibit kinase functions. GTP-RhoA binding relieves the auto-inhibition, switching-on the kinase activity . ROCKI and ROCKII have common substrates, such as myosin light chain (MLC), myosin binding subunit (MYPT1) of the MLC phosphatase, LIM kinases (LIMK1 and LIMK2), α-adducin, ezrin-radixin-moesin (ERM) proteins, and etc. [4, 5, 7–9]. Collectively, the kinase activation promotes the stabilization of actin filaments and myosin activity to increase actomyosin-driven cellular contractility [10, 11]. In addition to regulation by RhoA binding, ROCKs are negatively regulated by distinct binding proteins or phosphorylation. For example, Gem and RhoE specifically inhibit ROCKI and Rad for ROCKII inhibition [5, 12]. ROCKII has been shown to be phosphorylated at Tyr722 residue by Src kinase to decrease its affinity to GTP-RhoA , and dephosphorylated by Shp2 phosphatase . Therefore, the activity of ROCKI and II in vivo could be highly dependent on the cellular context.
To know the distinct biological roles of ROCKI and ROCKII, the ROCKI−/− and ROCKII−/− mice have been generated [15, 16]. ROCKI−/− mice are postnatal lethal, because of impairment of umbilical ring closure , and ROCKII−/− mice are embryonic lethal at the percentage of 90% due to the dysfunction of placenta and intrauterine growth retardation caused by thrombus formation in the labyrinth layer of placenta . These studies suggest that ROCKI and ROCKII have distinct functions in development.
Many reports have highlighted the association of ROCK activation with cancer progression and suggest the potential of ROCK as therapeutic targets in cancer [17–19]. The level of ROCKI RNA in tumor tissue correlates with the tumor grade and poor overall survival in breast cancer patients , and higher level of ROCKI protein has been found in osteosarcoma tissues . As for ROCKII, higher expression has been reported in aggressive hepatocellular carcinomas, colon and bladder cancers [22–24]. Considering that the expression level at mRNA or protein of ROCK may not be necessarily correlated with their kinase activity, we developed the reagents that can directly and specifically detect the activation status of ROCKI and ROCKII in cells and tissues by identification of their corresponding phosphorylation sites. Our previous results have provided evidence that ROCKII at Ser1366 residue reflects its kinase activation . In this study, we further showed activated ROCKI with phosphorylation at Ser1333 residue. Thus, the specific antibodies, one against ROCKI Ser1333 phosphorylation and another against ROCKII Ser1366 phosphorylation, can be used to detect the active form of ROCKI and ROCKII, respectively.
Plasmids and reagents
The S1333A mutation of ROCKI was introduced to wild-type pCMV2-flag-ROCKI described previously  using the Quick-Change site-directed mutagenesis kit (Stratagene). Y27632 was from Calbiochem-Novabiochem Corp.; λPPase was from New England Biolabs; nocodazole, anti-flag and anti-MLC antibodies were from Sigma-Aldrich; anti-ROCKI, anti-ROCKII and anti-RhoA antibodies were purchased from Santa Cruz Biotechnology; anti-phospho-MLC2 (T18/S19) antibody from Cell Signaling Technology; anti-pSer1366 ROCKII antibody was described previously .
Cell culture and transient transfection
Normal mouse embryonic fibroblasts (MEFs) and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2/95% air at 37°C. For transient transfection experiments, HEK293T cells were transfected by PolyJet reagent (SignaGen Laboratories).
Immunoprecipitation and in vitro kinase reaction
Flag-ROCKI-expressing cells were harvested in an IP buffer (1% NP-40, 5% glycerol, 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM PMSF, 50 mM NaF, 2 mM Na3VO4 and protease inhibitor cocktail). The lysates after pre-clearance were incubated with anti-flag antibody conjugated agarose beads (Sigma-Aldrich) at 4°C for 1 hr. The immunoprecipitates were pre-incubated with or without 100 μM of Y27632, which was followed by incubation with a kinase buffer (50 mM Tris–HCl, pH7.4, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 5 mM NaF, 0.1 mM Na3VO4, and 20 μM ATP) containing 5 μCi of [γ-32p]ATP at 30°C for 20 min. The reaction was stopped and products were separated by SDS-PAGE, transferred to a PVDF membrane. The phosphorylation status and amounts of the proteins were detected by autoradiography and Western blotting with anti-ROCKI antibody, respectively.
Phospho-specific antibody generation
The polyclonal anti-pS1333 ROCKI antibody was raised using phosphopeptide containing phosphorylated Ser1333 of ROCKI conjugated with keyhole limpet haemocyanin (KLH) as an antigen to immunize rabbits. Anti-sera were collected and sequentially affinity purified by phosphopeptide- and non-phosphopeptide-conjugated columns (ICON Biotechnology Co., Itd., Taiwan).
Results and discussion
Identification of activity-dependent phosphorylation site of ROCKI
Validation of Ser1333 phosphorylation of ROCKI by specific antibody
Detection of endogenous ROCKI activation by assessing Ser1333 phosphorylation
It is known that ROCKs form dimer [28–32]. In this study, we did not examine whether ROCKI S1333 phosphorylation is due to ROCKII in the heterodimer. It has been reported that the dimer consisting of wild-type kinase and catalytic-dead ROCK kinase domain is catalytically inactive . Therefore, we are unable to test whether ROCKII can phosphorylate catalytic-dead ROCKI at S1333 in the heterodimer form. In an overexpression experiment, we found that the amount of homodimer of ROCKII was more than 10-fold higher than that of ROCKI/II (data not shown). Considering that all ROCKs purified from a variety of tissues have been shown to be in homodimer form [2, 33–35], the physiological significance of S1333 phosphorylation in the heterodimer form of ROCKI/II is probably negligible. We also cannot exclude the possibility that other kinase is able to phosphorylate the S1333 site. Nevertheless, the correlation of S1333 phosphorylation with its upstream RhoA regulation and the extent of downstream substrate MLC phosphorylation suggest this modification as an indicator of ROCKI activation.
Small-molecule inhibitors against ROCK, such as Y27632 and Fasudil, have been developed to have potential in clinical implication [17, 36, 37]. Increasing number of clinical trials and animal experiments using these inhibitors suggest that ROCK activation plays an important role in the pathogenesis of many cardiovascular diseases, neurological disorders and cancers [3, 4, 38–43]. Although the functions of ROCKI and ROCKII are analogous and compensatory, genetic deletion studies suggest that each kinase might play distinct roles depending on tissue types and certain biological processes. Also unknown is which ROCK isoform is responsible for pathogenesis of a specific tissue in diseases and related to disease progression. Our antibodies that can detect the phosphorylation of ROCKI at Ser1333 and ROCKII at Ser1366 offer new opportunities to differentiate the activation status of ROCKI and II in association with diseases. Of note, all the current inhibitors cannot discern between ROCKI and II. The antibodies that can detect active forms of ROCKI and II provide valuable tools for screening ROCKI and ROCKII specific inhibitors.
Ser1333 phosphorylation can indicate the active status of ROCKI in response to RhoA signaling. Thus, antibodies that recognize phosphorylation at Ser1333 and S1366 residues of ROCKI and II, respectively, are capable of probing their corresponding activation in biological samples. Also, these antibodies might be very useful reagents for drug screening of inhibitors specific against ROCKI and ROCKII isoform.
This research is supported by Taiwan National Science Council [grant number NSC102-2628-B-010-003-MY3], and in part by the UST-UCSD International Center of Excellence in Advanced Bio-engineering sponsored by the Taiwan National Science Council I-RiCE Program [grant number NSC-102-2911-I-009-101]. We also are grateful for the support from the Ministry of Education in National Yang-Ming University, Aim for the Top University Plan.
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