The PDZ-LIM protein RIL modulates actin stress fiber turnover and enhances the association of α-actinin with F-actin☆
Introduction
The major role of the actin cytoskeleton in muscle cells is to act as contractile machinery, whereas in nonmuscle cells, it is involved in multiple processes including cell polarity, cell shape change, motility and cytokinesis. To perform all of these tasks, the nonmuscle actin cytoskeleton is composed of a complex set of proteins that interact with different signaling pathways. The exact composition of the actin cytoskeleton and the signaling pathways interacting with it are only partially characterized.
Several recently identified PDZ-LIM proteins have been suggested to participate in cytoskeletal functions in muscle and nonmuscle cells. These proteins contain one N-terminal PDZ domain followed either by one C-terminal LIM domain (ALP subfamily; ALP, RIL, CLP-36) or three C-terminal LIM domains (Enigma subfamily; Enigma, ENH, ZASP/Cypher1) (Ref. [1], and therein). It has been suggested that PDZ-LIM proteins act as adapters recruiting signaling molecules to the actin cytoskeleton [2]. This is based on the ability of some PDZ-LIM proteins to associate with the actin cytoskeleton via their PDZ domains [2], [4], and also on the ability of several PDZ-LIM proteins to associate with kinases via their LIM domains [5], [6], [7], [8], [9].
The muscle-specific ALP and Cypher1 are implicated in normal sarcomere function. Both are localized to Z-discs in sarcomeres, where they associate with α-actinin [1], [9], a major component of Z-discs. Gene ablation experiments in mice demonstrated that Alp−/− mice gradually develop cardiomyopathy [10], and Cypher1−/− mice die postnatally with a severe form of congenital myopathy [11]. Interestingly, ultrastructural analysis of Cypher1−/− muscle tissue indicates that the initial formation of the Z-disc is normal but soon after contraction, Z-disc structures become disorganized and disrupted, suggesting that Cypher1 is required specifically for maintenance of the Z-disc [11]. Mice deficient for another Z-disc protein MLP have similar phenotypes leading to the suggestion that some Z-disc proteins might function as a sensor of biomechanical stress [12], [13]. Although the mechanism of this sensor remains to be characterized, the interaction between the PDZ domain of Cypher1 and/or ALP with α-actinin [1], [9] as well as the association of Cypher1 LIM domain with protein kinase C [9] are likely to be involved.
The predominantly nonmuscle CLP-36 PDZ-LIM protein was recently demonstrated to associate with actin stress fibers in nonmuscle cells [4], [14]. Stress fibers are sarcomere-like structures with several shared components including myosin, tropomyosin, titin and α-actinin [15], [16], [17], [18]. Stress fibers are also contractile elements [19], which contract, for example, in response to restimulation of serum-deprived Swiss 3T3 cells [20]. Stress fiber contraction has been suggested to activate myosin II, leading to increased cell stiffness and decreased cell spreading [21]. Thus actin stress fiber dynamics and turnover are critically involved in actin cytoskeleton functions.
The third characterized member of the ALP subfamily of PDZ-LIM protein, RIL, was identified initially as a gene downregulated in H-ras-transformed cells [22], and later also as a protein interacting with the PTP-BL phosphatase [23]. In this study, we have studied the expression and functions of RIL in nonmuscle cells especially with regard to similarities and differences with CLP-36.
Section snippets
In situ hybridization
Embryos at stages E7, E9, E11, E14, E17.5 of NMRI mice were timed by both vaginal plugs of mothers and by morphological criteria. Mouse antisense Ril, Clp-36 and Alp RNA probes were synthesized from 440 bp (NM_019417, nucleotides 313–752), 825 bp (AF053367, nucleotides 158–982) and 323 bp (NM_016798, nucleotides 1035–1357) PCR fragments, and subloned into pGEM-T vector (Promega). Tissue preparation and in situ hybridization with these probes were performed as described [24].
Cell culture and antibodies
U2OS osteosarcoma
Distinct expression patterns of Ril and Clp-36
ALP, RIL and CLP-36 form a clearly defined subfamily of PDZ-LIM proteins with a similar domain structure, a similar overall length and high sequence similarity (Fig. 1A). The overall homology is slightly higher between RIL and CLP-36 (43% identity) than that between RIL and ALP (41% identity), whereas CLP-36 and ALP share the highest homology with each other (50% identity). Expression of ALP has been reported to be restricted to muscle [1], [27], whereas CLP-36 and RIL are expressed also in
Discussion
The RIL PDZ-LIM protein together with ALP and CLP-36 form the ALP subfamily of PDZ-LIM proteins. Previous studies have reported several similarities between CLP-36 and ALP, including localization to actin filaments, and association with α-actinin via their PDZ domains [1], [4]. Results presented here indicate that RIL mimics CLP-36 both in its localization to actin stress fibers and in its PDZ-mediated association with α-actinin. However, our results also demonstrate several important
Acknowledgements
We thank Birgitta Tjäder and Sanna Kihlberg for technical assistance and Päivi Ojala and the Mäkelä laboratory for scientific discussions. This study has been supported by grants from The Finnish Medical Foundation, Farmos Research Foundation, the Emil Aaltonen Foundation, Ida Montinin Foundation, Biocentrum Helsinki, Finnish Cancer Organization, Sigrid Juselius Foundation and the Academy of Finland. T.V. is a student of the Helsinki Biomedical Graduate School.
References (32)
- et al.
CLP-36 PDZ-LIM protein associates with nonmuscle alpha-actinin-1 and alpha-actinin-4
J. Biol. Chem.
(2000) - et al.
Mitogenic signaling by Ret/ptc2 requires association with enigma via a LIM domain
J. Biol. Chem.
(1996) - et al.
Protein–protein interaction of zinc finger LIM domains with protein kinase C
J. Biol. Chem.
(1996) - et al.
Specificity of LIM domain interactions with receptor tyrosine kinases
J. Biol. Chem.
(1996) - et al.
Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to alpha-actinin-2 and protein kinase C
J. Biol. Chem.
(1999) The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy
Cell
(2002)Human CLP36, a PDZ-domain and LIM-domain protein, binds to alpha-actinin-1 and associates with actin filaments and stress fibers in activated platelets and endothelial cells
Blood
(2000)- et al.
Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells
Cell
(1975) - et al.
Stress fiber sarcomeres of fibroblasts are contractile
Cell
(1980) - et al.
Purification and characterization of an alpha-actinin-binding PDZ-LIM protein that is up-regulated during muscle differentiation
J. Biol. Chem.
(1999)
The zyxin-related protein TRIP6 interacts with PDZ motifs in the adaptor protein RIL and the protein tyrosine phosphatase PTP-BL
Eur. J. Cell Biol.
The human TRIP6 gene encodes a LIM domain protein and maps to chromosome 7q22, a region associated with tumorigenesis
Genomics
Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat
J. Cell Biol.
The PDZ domain of the LIM protein enigma binds to beta-tropomyosin
Mol. Biol. Cell
Clik1: a novel kinase targeted to actin stress fibers by the CLP-36 PDZ-LIM protein
J. Cell Sci.
Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy
Nat. Med.
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Supplementary data associated with this article can be found, in the online version, at doi 10.106/S0014-4827(03)00484-1.
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Present address: Merck KGaA, Frankfurter Str. 250, D-64293, Darmstadt, Germany.