Ca2+-sensitive tyrosine kinase Pyk2/CAK β-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy

doi:10.1016/j.yjmcc.2004.03.002

Journal of Molecular and Cellular Cardiology

Volume 36, Issue 6, June 2004, Pages 799-807

https://doi.org/10.1016/j.yjmcc.2004.03.002 Get rights and content

Abstract

G-protein-coupled receptor agonists including endothelin-1 (ET-1) and phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Others and we previously reported that Rac1 signaling pathway plays an important role in this agonist-induced cardiomyocyte hypertrophy. In this study reported here, we found that a Ca²⁺-sensitive non-receptor tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase β (CAKβ), is involved in ET-1- and PE-induced cardiomyocyte hypertrophy medicated through Rac1 activation. ET-1, PE or the Ca²⁺ inophore, ionomycin, stimulated a rapid increase in tyrosine phosphorylation of Pyk2. The tyrosine phosphorylation of Pyk2 was suppressed by the Ca²⁺ chelator, BAPTA. ET-1- or PE-induced increases in [³H]-leucine incorporation and expression of atrial natriuretic factor and the enhancement of sarcomere organization. Infection of cardiomyocytes with an adenovirus expressing a mutant Pyk2 which lacked its kinase domain or its ability to bind to c-Src, eliminated ET-1- and PE-induced hypertrophic responses. Inhibition of Pyk2 activation also suppressed Rac1 activation and reactive oxygen species (ROS) production. These findings suggest that the signal transduction pathway leading to hypertrophy involves Ca²⁺-induced Pyk2 activation followed by Rac1-dependent ROS production.

Introduction

Cardiomyocytes are regarded as terminally differentiated cells, in which adaptive hypertrophic growth in response to a variety of extracellular stimuli involves an increase in protein content and cell size, changes in myofibrillar organization and gene expression. Analysis of the signal transduction cascade in the development of cardiac hypertrophy has used neonatal ventricular myocytes as a model system. The signaling pathways, in which extracellular signals are transmitted into myocytes to induce the hypertrophic responses, are probably multifold. The small (21-kDa) guanine nucleotide-binding protein, Rac has been implicated in cardiac hypertrophic responses, mediating both the morphological and transcriptional changes [1], [2], [3]. Previous studies have demonstrated that hypertrophic G-protein-coupled receptor (GPCR) agonists such as endothelin-1 (ET-1) and phenylephrine (PE) induce rapid activation of Rac1 in cardiomyocytes [2]. Adenoviral gene transfer of a constitutively active mutant of Rac1 resulted in enhancement of sarcomeric organization and an increase in cell size and protein synthesis along with an increase in atrial natriuretic factor (ANF) expression, whereas infection with an adenovirus expressing a dominant-negative mutant of Rac1 inhibited the PE-induced hypertrophic responses [3], [4]. Rac1 induces cardiomyocyte hypertrophy in reactive oxygen species (ROS)-dependent manner [3], [5]. However, the signaling molecules upstream of Rac1 have to be elucidated.

Proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase β (CAKβ), also known as related adhesion focal tyrosine kinase or Ca²⁺-dependent tyrosine kinase, is related to focal adhesion kinase (FAK) and is regulated by several intracellular stimuli [6], [7], [8], [9]. Pyk2 is activated by hormones, GPCR agonists, membrane depolarization and an increase in intracellular Ca²⁺ [7], [10]. Pyk2 has been implicated in the regulation of ion channels [7], cell adhesion and motility [11].

Pyk2 is expressed in cardiomyocytes and hypertrophic agonists such as ET-1 induce the phosphorylation of Pyk2 [12], [13]. Furthermore, Pyk2 expression and phosphorylation increase in pressure overload-induced cardiac hypertrophy [14]. These findings suggest that Pyk2 may play a role in cardiac hypertrophy. Involvement of Pyk2 in cardiomyocyte hypertrophy, however, remains to be clarified. Inhibition of Pyk2 or Rac1 abolished angiotensin II-induced c-Jun NH₂-terminal kinase (JNK) activation in cardiac fibroblasts, suggesting Pyk2 and Rac1 may share a common signaling pathway [15].

In this study, we examined whether Pyk2 is involved in cardiomyocyte hypertrophy. We also explored the possibility that the presence of Rac1 downstream of Pyk2 leads to cardiac myocyte hypertrophy. We demonstrated that Pyk2 mediates ET-1- and PE-induced cardiomyocyte hypertrophy via Rac1 activation.

Section snippets

Cardiomyocyte culture and adenoviral infection

Rat ventricular myocytes from 1 to 2-d-old Wister rats were prepared as described previously [16]. Cardiomyocytes were plated in serum-containing medium (Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 400 units/ml penicillin, 200 units/ml streptomycin) overnight. Subsequently, the cells were incubated in a non-serum medium containing 5 μg/ml transferrin and 1 μg/ml insulin and were infected with adenoviruses at a multiplicity of infection of 10–20 particles/cell for 24 h.

ET-1 and PE stimulate tyrosine phosphorylation of Pyk2 in cardiomyocytes

It has been reported that ET-1-induced phosphorylation of Pyk2 in neonatal cardiomyocytes [12], [13]. We examined whether another GPCR agonist, PE also activates Pyk2 in neonatal cardiomyocytes. After exposure to ET-1 or PE, Pyk2 was immunoprecipitated with the anti-Pyk2 antibody, and the phosphorylation level of Pyk2 was measured by using the anti-tyrosine antibody. Treatment with ET-1 or PE significantly was found to increase phosphorylation of Pyk2 (Fig. 1A,B). The increase started within

Discussion

In this study, we found evidences that GPCR agonists induce the activation of Pyk2 in cardiomyocytes, in that inhibition of Pyk2 attenuated all features of GPCR agonist-induced hypertrophic responses including increases in protein synthesis and sarcomere organization and reactivation of fetal genes such as ANF. To confirm the involvement of Pyk2 in cardiomyocyte hypertrophy, it has to be demonstrated that activated Pyk2 can lead to cardiomyocyte hypertrophy. To the best of our knowledge,

Acknowledgements

We wish to thank Ms Ritsuko Okamoto and Atsuko Nakai for their excellent technical assistance. This work was supported by Grant-in-Aids from the Ministry of Education, Culture and Science, Japan (to K.O.).

References (32)

Y Higuchi et al.
The small GTP-binding protein Rac1 induces cardiac myocyte hypertrophy through the activation of apoptosis signal-regulating kinase 1 and NF-kappa B
J Biol Chem
(2003)
H Sasaki et al.
Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily
J Biol Chem
(1995)
S Avraham et al.
Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain
J Biol Chem
(1995)
M.D Schaller et al.
Differential signaling by the focal adhesion kinase and cell adhesion kinase beta
J Biol Chem
(1997)
H Yu et al.
Activation of a novel calcium-dependent protein-tyrosine kinase
J Biol Chem
(1996)
A Astier et al.
The related adhesion focal tyrosine kinase differentially phosphorylates p130Cas and the Cas-like protein, p105HEF1
J Biol Chem
(1997)
A.L Bayer et al.
Pyk2 expression and phosphorylation in neonatal and adult cardiomyocytes
J Mol Cell Cardiol
(2001)
H Kodama et al.
Role of EGF receptor and Pyk2 in endothelin-1-induced ERK activation in rat cardiomyocytes
J Mol Cell Cardiol
(2002)
S Murasawa et al.
Angiotensin II initiates tyrosine kinase Pyk2-dependent signalings leading to activation of Rac1-mediated c-Jun NH2-terminal kinase
J Biol Chem
(2000)
H Tang et al.
Pyk2/CAKbeta tyrosine kinase activity-mediated angiogenesis of pulmonary vascular endothelial cells
J Biol Chem
(2002)

G.D Frank et al.

PYK2/CAK[beta] represents a redox-sensitive tyrosine kinase in vascular smooth muscle cells

Biochem Biophys Res Commun

(2000)

J Melendez et al.

Activation of pyk2/related focal adhesion tyrosine kinase and focal adhesion kinase in cardiac remodeling

J Biol Chem

(2002)

A Blaukat et al.

Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades

J Biol Chem

(1999)

X Li et al.

Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase

J Biol Chem

(1999)

S.J Fuller et al.

Oncogenic src, raf, and ras stimulate a hypertrophic pattern of gene expression and increase cell size in neonatal rat ventricular myocytes

J Biol Chem

(1998)

L.M Graves et al.

An intracellular calcium signal activates p70 but not p90 ribosomal S6 kinase in liver epithelial cells

J Biol Chem

(1997)

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Original ArticleCa2+-sensitive tyrosine kinase Pyk2/CAK β-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy

Abstract

Introduction

Section snippets

Cardiomyocyte culture and adenoviral infection

ET-1 and PE stimulate tyrosine phosphorylation of Pyk2 in cardiomyocytes

Discussion

Acknowledgements

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Mol Cell Cardiol

J Mol Cell Cardiol

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Original Article
Ca²⁺-sensitive tyrosine kinase Pyk2/CAK β-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy