Proteomic Studies of PP2A-B56γ1 Phosphatase Complexes Reveal Phosphorylation-Regulated Partners in Cardiac Local Signaling

Zhou, Xing; Mudannayake, Malkanthi; Green, Mariah; Gigena, Marisa S.; Wang, Guanghui; Shen, Rong-Fong; Rogers, Terry B.

doi:10.1021/pr060619l

Cited by 10 publications

(6 citation statements)

References 60 publications

(121 reference statements)

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“…In Vitro Phosphorylation Assay-The His-tagged full-length Smad1 was expressed in Escherichia coli BL21 (DE3) strain and purified by using Ni 2ϩ -Sepharose beads (Qiagen) as described previously (37). The GST-tagged ROP18 ⌬83 and ROP18 ⌬83 -KD recombinant proteins were purified as described above.…”

Section: Methodsmentioning

confidence: 99%

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18

Yang

Hou

Hao

et al. 2017

Molecular & Cellular Proteomics

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Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface. Molecular &

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Section: Methodsmentioning

confidence: 99%

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18

Yang

Hou

Hao

et al. 2017

Molecular & Cellular Proteomics

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“…The principles governing the care and treatment of animals as described by the American Physiological Society were followed at all times during this study. Isolation of adult rat ventricular myocytes (ARVM) was performed by Langendorff perfusion with a buffer containing low Ca 2+ , collagenase and protease as described in our previous papers [9, 10]. Isolation and culture of neonatal rat ventricular myocytes (NRVM) were conducted using the overnight trypsin-collagenase digestion method as described [9, 10].…”

Section: Methodsmentioning

confidence: 99%

“…Isolation of adult rat ventricular myocytes (ARVM) was performed by Langendorff perfusion with a buffer containing low Ca 2+ , collagenase and protease as described in our previous papers [9, 10]. Isolation and culture of neonatal rat ventricular myocytes (NRVM) were conducted using the overnight trypsin-collagenase digestion method as described [9, 10]. All experiments with NRVMs were performed on 2–4 d cultures when synchronously contracting cells were observed.…”

Section: Methodsmentioning

confidence: 99%

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

Cai

Sun

et al. 2011

Biochemical and Biophysical Research Communications

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Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are 3 δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and β-myocin heavy chain (β-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target.

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“…One of the four unrelated gene families of the B subunit is B′ (or B56). Its members are encoded by five distinct genes, generating a variety of splicing isoforms, and are preferentially expressed in cardiac muscle (Ito et al 2003; Zhou et al 2007). Although there is growing knowledge about the structural properties of PP2A subunits, there is little information on their distribution and functional interplay in normal and diseased myocardium.…”

Section: Introductionmentioning

confidence: 99%

Protein phosphatase 2A affects myofilament contractility in non-failing but not in failing human myocardium

Wijnker

Boknı́k

Gergs

et al. 2011

J Muscle Res Cell Motil

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Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation–contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50 = 0.05 ± 0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50 = 0.10 ± 0.03) and dilated (DCM, ΔpCa50 = 0.06 ± 0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts.

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Proteomic Studies of PP2A-B56γ1 Phosphatase Complexes Reveal Phosphorylation-Regulated Partners in Cardiac Local Signaling

Cited by 10 publications

References 60 publications

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

Protein phosphatase 2A affects myofilament contractility in non-failing but not in failing human myocardium

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