Zipper-interacting Protein Kinase (ZIPK) Modulates Canonical Wnt/β-Catenin Signaling through Interaction with Nemo-like Kinase and T-cell Factor 4 (NLK/TCF4)

As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK(1–320)-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC20 and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility.

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Zipper interacting protein kinase (ZIPK): function and signaling

APOPTOSIS ◽

10.1007/s10495-013-0934-3 ◽

2013 ◽

Vol 19 (2) ◽

pp. 387-391 ◽

Cited By ~ 19

Author(s):

Tatsuya Usui ◽

Muneyoshi Okada ◽

Hideyuki Yamawaki

Keyword(s):

Protein Kinase ◽

Interacting Protein ◽

Zipper Interacting Protein Kinase

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Protein Kinase D1 attenuates tumorigenesis in colon cancer by modulating β-catenin/T cell factor activity

Oncotarget ◽

10.18632/oncotarget.2277 ◽

2014 ◽

Vol 5 (16) ◽

pp. 6867-6884 ◽

Cited By ~ 10

Author(s):

Vasudha Sundram ◽

Aditya Ganju ◽

Joshua E. Hughes ◽

Sheema Khan ◽

Subhash C. Chauhan ◽

...

Keyword(s):

Colon Cancer ◽

T Cell ◽

Protein Kinase ◽

Factor Activity ◽

Protein Kinase D1 ◽

T Cell Factor

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Sox17 and Sox4 Differentially Regulate β-Catenin/T-Cell Factor Activity and Proliferation of Colon Carcinoma Cells

Molecular and Cellular Biology ◽

10.1128/mcb.02179-06 ◽

2007 ◽

Vol 27 (22) ◽

pp. 7802-7815 ◽

Cited By ~ 190

Author(s):

Débora Sinner ◽

Jennifer J. Kordich ◽

Jason R. Spence ◽

Robert Opoka ◽

Scott Rankin ◽

...

Keyword(s):

T Cell ◽

Wnt Signaling ◽

Colon Carcinoma ◽

Carcinoma Cells ◽

Loss Of Function ◽

T Cell Factor ◽

Colon Carcinoma Cells ◽

Intestinal Neoplasia ◽

Sox Proteins ◽

Canonical Wnt

ABSTRACT The canonical Wnt pathway is necessary for gut epithelial cell proliferation, and aberrant activation of this pathway causes intestinal neoplasia. We report a novel mechanism by which the Sox family of transcription factors regulate the canonical Wnt signaling pathway. We found that some Sox proteins antagonize while others enhance β-catenin/T-cell factor (TCF) activity. Sox17, which is expressed in the normal gut epithelium but exhibits reduced expression in intestinal neoplasia, is antagonistic to Wnt signaling. When overexpressed in SW480 colon carcinoma cells, Sox17 represses β-catenin/TCF activity in a dose-dependent manner and inhibits proliferation. Sox17 and Sox4 are expressed in mutually exclusive domains in normal and neoplastic gut tissues, and gain- and loss-of-function studies demonstrate that Sox4 enhances β-catenin/TCF activity and the proliferation of SW480 cells. In addition to binding β-catenin, both Sox17 and Sox4 physically interact with TCF/lymphoid enhancer factor (LEF) family members via their respective high-mobility-group box domains. Results from gain- and loss-of-function experiments suggest that the interaction of Sox proteins with β-catenin and TCF/LEF proteins regulates the stability of β-catenin and TCF/LEF. In particular, Sox17 promotes the degradation of both β-catenin and TCF proteins via a noncanonical, glycogen synthase kinase 3β-independent mechanism that can be blocked by proteasome inhibitors. In contrast, Sox4 may function to stabilize β-catenin protein. These findings indicate that Sox proteins can act as both antagonists and agonists of β-catenin/TCF activity, and this mechanism may regulate Wnt signaling responses in many developmental and disease contexts.

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Zipper-interacting protein kinase is involved in regulation of ubiquitination of the androgen receptor, thereby contributing to dynamic transcription complex assembly

Oncogene ◽

10.1038/onc.2012.503 ◽

2012 ◽

Vol 32 (41) ◽

pp. 4981-4988 ◽

Cited By ~ 8

Author(s):

A Felten ◽

D Brinckmann ◽

G Landsberg ◽

K H Scheidtmann

Keyword(s):

Androgen Receptor ◽

Protein Kinase ◽

Complex Assembly ◽

Interacting Protein ◽

Zipper Interacting Protein Kinase

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Zipper‐interacting protein kinase is a key regulator of vascular smooth muscle tone with implications in development of hypertension (676.18)

The FASEB Journal ◽

10.1096/fasebj.28.1_supplement.676.18 ◽

2014 ◽

Vol 28 (S1) ◽

Author(s):

Sara Turner ◽

Timothy Haystead ◽

Justin MacDonald

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Vascular Smooth Muscle ◽

Muscle Tone ◽

Interacting Protein ◽

Smooth Muscle Tone ◽

Zipper Interacting Protein Kinase ◽

Vascular Smooth Muscle Tone

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Testosterone Inhibits Adipogenic Differentiation in 3T3-L1 Cells: Nuclear Translocation of Androgen Receptor Complex with β-Catenin and T-Cell Factor 4 May Bypass Canonical Wnt Signaling to Down-Regulate Adipogenic Transcription Factors

Endocrinology ◽

10.1210/en.2004-1649 ◽

2006 ◽

Vol 147 (1) ◽

pp. 141-154 ◽

Cited By ~ 252

Author(s):

Rajan Singh ◽

Jorge N. Artaza ◽

Wayne E. Taylor ◽

Melissa Braga ◽

Xin Yuan ◽

...

Keyword(s):

Androgen Receptor ◽

T Cell ◽

Wnt Signaling ◽

Fat Mass ◽

Nuclear Translocation ◽

Adipogenic Differentiation ◽

Inhibitory Effects ◽

T Cell Factor ◽

Enhancer Binding Protein ◽

Canonical Wnt

Testosterone supplementation in men decreases fat mass; however, the mechanisms by which it inhibits fat mass are unknown. We hypothesized that testosterone inhibits adipogenic differentiation of preadipocytes by activation of androgen receptor (AR)/β-catenin interaction and subsequent translocation of this complex to the nucleus thereby bypassing canonical Wnt signaling. We tested this hypothesis in 3T3-L1 cells that differentiate to form fat cells in adipogenic medium. We found that these cells express AR and that testosterone and dihydrotestosterone dose-dependently inhibited adipogenic differentiation as analyzed by Oil Red O staining and down-regulation of CCAAT/enhancer binding protein-α and -δ and peroxisome proliferator-activated receptor-γ2 protein and mRNA. These inhibitory effects of androgens were partially blocked by flutamide or bicalutamide. Androgen treatment was associated with nuclear translocation of β-catenin and AR. Immunoprecipitation studies demonstrated association of β-catenin with AR and T-cell factor 4 (TCF4) in the presence of androgens. Transfection of TCF4 cDNA inhibited adipogenic differentiation, whereas a dominant negative TCF4 cDNA construct induced adipogenesis and blocked testosterone’s inhibitory effects. Our gene array analysis indicates that testosterone treatment led to activation of some Wnt target genes. Expression of constitutively activated AR fused with VP-16 did not inhibit the expression of CCAAT/enhancer binding protein-α in the absence of androgens. Testosterone and dihydrotestosterone inhibit adipocyte differentiation in vitro through an AR-mediated nuclear translocation of β-catenin and activation of downstream Wnt signaling. These data provide evidence for a regulatory role for androgens in inhibiting adipogenic differentiation and a mechanistic explanation consistent with the observed reduction in fat mass in men treated with androgens.

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Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase

Biochemical Journal ◽

10.1042/bj20020401 ◽

2002 ◽

Vol 366 (1) ◽

pp. 211-216 ◽

Cited By ~ 132

Author(s):

Andrea MURÁNYI ◽

Justin A. MacDONALD ◽

Jing Ti DENG ◽

David P. WILSON ◽

Timothy A.J. HAYSTEAD ◽

...

Keyword(s):

Protein Kinase ◽

Rho Kinase ◽

Inhibitory Effect ◽

Myosin Phosphatase ◽

Western Blots ◽

Interacting Protein ◽

Terminal Fragment ◽

Inhibitory Site ◽

Zipper Interacting Protein Kinase ◽

Integrin Linked Kinase

A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr709, and two other sites at Thr695 and Thr495. One of the sites for cAMP-dependent protein kinase (PKA) was Ser694. Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr695→Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr695 was the major inhibitory site, Thr709 had only a slight inhibitory effect and Ser694 had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.

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