The actin cytoskeleton undergoes major changes during T lymphocyte migration, antigen-driven activation, apoptosis, and infection with retroviruses [10
]. These events result in the activation of various signaling components that are assembled and brought into the close proximity by the actin cytoskeleton. Actin cytoskeletal homeostasis also regulates gene expression by modulating chromatin remodeling, mRNA processing, and nuclear export [24
]. Thus actin pathways regulate cellular functions by the dual processes of modulating signaling and also directly participating in nuclear transcription. It is known that retinoid receptor function is attenuated during T cell activation [6
], a phenomenon that involves actin remodeling, suggesting that actin modification may play a role in such inhibition. In this study we set out to investigate the role of actin cytoskeleton homeostasis and dynamics in retinoid receptor-mediated transcription. Latrunculin A, swinholide A, compounds that prevent F-actin formation, or jasplakinolide, a chemical that induces F-actin formation and stabilization, were all found to attenuate transcription to various levels. These results point out the importance of actin homeostasis in retinoid receptor-mediated activation and indicate that any disturbances in actin dynamics and the F-actin homeostasis attenuate retinoid receptor-dependent transcription. Electrophoretic mobility shift assay did not reveal any significant loss of RARE or RXRE probe binding activity in response to F-actin modifying agent (data not shown) suggesting that mechanisms other than the effect on DNA binding are involved in transcriptional inhibition induced by F-actin disruption.
CFL1 is a key F-actin remodeling protein that severs F-actin and thereby allows addition and assembly of new actin filaments, the so-called actin-treadmilling [15
]. Both siRNA-mediated knockdown and overexpression of CFL1 attenuated retinoid receptor function showing that CFL1 homeostasis plays a critical role in receptor activity. This was further evident from the data obtained with constitutively active (S3A) and inactive/dominant-negative (S3E) mutants of CFL1. Whereas the transfection of cells with CFL1/WT and S3A mutant induced loss of F-actin content in the cells expressing these proteins, transfection with S3E mutant increased F-actin content consistent with the ability of WT and S3A mutant to severe F-actin and S3E mutant to increase F-actin content. Loss of transcriptional activation by the expression of S3A and S3E mutants of CFL1, in addition to CFL1/WT, further emphasizes the importance of actin homeostasis in the regulation of retinoid receptor-mediated transcription.
Interestingly, CFL1 overexpression has also been shown to negatively regulate glucocorticoid receptor function [31
] and thrombin-induced NF-kappaB activity [37
] suggesting that CFL1 homeostasis is also essential for the activities of steroid hormone-mediated nuclear receptors as well as other transcription factors besides retinoid receptors reported in this study.
The role of LIMK1 in the regulation of nuclear hormone receptor-dependent activation is unknown. Here we have shown that when overexpressed LIMK1 functions as a negative regulator of retinoid receptor function. The expression of an enzymatically inactive D460A mutant of LIMK1, that also functions as a dominant-negative protein, also inhibited receptor activity. These data suggest that physiological levels of LIMK1 are crucial for receptor function due to its ability to regulate CFL1 and actin cytoskeletal homeostasis. Overexpression of LIMK1 and loss of LIMK1 activity (D460A mutant expression) increased and decreased F-actin content respectively, and deregulated actin dynamics and homeostasis so crucial for retinoid receptor activity.
A number of previous studies have demonstrated that Nef expression disturbs F-actin cytoskeleton [17
]. We confirmed these observations in this study and found that Nef expression induced loss of the characteristic actin ring structure. In addition, we report that Nef inhibited retinoid receptor mediated transcription. The gene array analysis of Nef-transfected 293 cells identified inhibition of endogenous expression of a number of genes that contain retinoid receptor binding sites in their promoters. IFIT1, CEBPB, and CRABP2 are known retinoic acid inducible genes [34
] whereas RBM8A and ACOT2 were found to contain DR2 (RARE) and DR1 (RXRE) consensus sequence elements, respectively, within their promoter regions upstream of the transcription start site.
The role of myristoylation in reducing the receptor function correlated well with the inability of this Nef mutant to induce loss of F-actin ring structure. The conclusion that the Nef-mediated loss of transcriptional activation is a consequence of disturbance in the actin dynamics was supported by the observation that the presence of Nef exhibited a cooperative effect on the loss of transcription induced by the actin-modifying agents. These data show that retinoid receptor mediated transcription is tightly controlled by actin cytoskeletal homeostasis and any disturbances in F-actin dynamics that may include changes in F-actin architecture, length, and content affect receptor-driven transcription.
In order to gain further insight to understand the role of actin pathways in modulating retinoid receptor-mediated function, we next studied the mechanism by which Nef inhibits actin dynamics. We have shown that cells expressing Nef had higher levels of phospho-CFL1 suggesting a link between Nef mediated phosphorylation of CFL1 and inhibition of retinoid receptor-mediated activation. By inhibiting CFL1 activity Nef protein disturbs F-actin homeostasis that is crucial for normal retinoid receptor function. Nef induced phosphorylation of CFL1 has also recently been reported by others [20
] indicating that modification of CFL1 activity is an important mechanism by which Nef modulates actin-cytoskeletal dynamics and cellular functions. The mechanism underlying the phosphorylation of CFL1 by Nef is not known. Although phospho-CFL1 appears to colocalize with Nef (Figure ), but Nef did not interact with CFL1 in immunoprecipitation experiments (unpublished observation). Since Nef lacks any kinase activity, phosphorylation of CFL1 by Nef is probably induced due its activation and association with other kinases and/or adapter proteins like VAV. Nef has been reported to associate with p-21 associated kinases (PAK) 1 and 2 and induce activation of these serine/threonine kinases [39
]. PAKs are effectors of RAC and CDC42 GTPases that are known for their role in actin dynamics. PAK1 is an upstream kinase for LIMK1 that binds and activates LIMK1 by phosphorylating Thr508 within the activation loop, resulting in a marked increase in its activity to phosphorylate CFL1 [42
]. In fact treatment of Jurkat cells with PAK inhibitor IPA3 was able to induce retinoid receptor transcription and reduce p-CFL1 levels (data not shown) indicating that PAK-mediated CFL1 phosphocycling plays an important role in retinoid receptor function.
The ability of Nef to inhibit transcription and induce CFL1 phosphorylation suggested that Nef might function by activating LIMK1 thereby inducing CFL1 phosphorylation. Data presented here support this hypothesis and demonstrated that the expression of myristoylated Nef induced significant phosphorylation of LIMK1 at Thr508. How Nef induces phosphorylation of LIMK1 is not known but Nef-mediated activation of PAK1 that is known to directly phosphorylate LIMK1 [42
] remains a possibility. We also found that expression of Nef induces the "rounding off" phenotype in cells that is indicative of cell cycle changes seen in mitotic cells [43
]. This rounding phenotype induced by Nef was associated with increased phospho-CFL1 and phospho-LIMK1 levels (Figures and ). Although a number of previous studies have shown that there is a transient net change in the phospho-CFL1 levels and increase in the phospho-LIMK1 levels during mitosis and cytokinesis [45
], but our cell cycle studies did not reveal any significant changes in the number of cells in mitotic phase in cells transfected with Nef (data not shown). It is possible that the "rounding off" phenotype seen in this study may reflect the morphological changes in cells due to disturbance in F-actin dynamics and not the presence of mitotic cells.
Our data strongly indicates that Nef is a modulator of LIMK1 and CFL1 phosphorylation that modifies the function of these two proteins and inhibits retinoid receptor transcription by interfering in actin architecture. This is further emphasized in our overexpression data using various CFL1 and LIMK1 mutants that induce loss of transcription and disturbance in the actin dynamics. Data obtained with Nef protein not only confirm the role of actin dynamics in retinoid receptor transcription, but also provide mechanistic information. Although we cannot rule out the possibility that HIV-Nef inhibits receptor function through additional mechanisms besides modifying CFL1 phosphocycling, modification of actin dynamics by Nef and the crucial role of actin homeostasis in receptor function provide evidence that Nef inhibits receptor function largely by inhibiting actin dynamics.
In conclusion, we have described a critical role of actin-cytoskeleton dynamics in normal retinoid receptor-mediated function and found that LIMK1-mediated phosphocycling of CFL1 plays a crucial role in maintaining actin homeostasis and receptor activity. Our studies however, do not rule out the importance of LIMK2 in retinoid receptor function. We have identified HIV-1 Nef protein as an inhibitor of receptor function by virtue of its LIMK1-meditaed CFL1 modifying activity and used it as tool to study the importance of actin dynamics in receptor function. Further studies are needed to identify the mechanism by which disturbances in actin dynamics directly alter the ability of retinoid receptors to activate transcription in the nuclear compartment. A number of studies have demonstrated that nuclear actin exists in dynamic equilibrium between monomeric and polymeric forms, indicating that polymerization of monomeric G-actin is an essential feature of actin dynamics in the nucleoplasm [49
]. A recent report has shown that most of the G-actin pool in the nucleus is bound to CFL suggesting the importance of CFL in the regulation of nuclear function [51
]. LIMK1 [45
] and CIN are known to have nuclear functions [47
CFL1 is an essential component of T cell activation process and is crucial for IS formation. Most of the CFL1 in naive T cells is found in the inactive phosphorylated form that following TCR activation, is dephosphorylated into an active form [10
]. Our previous studies have revealed that retinoid receptor function is inhibited during T cell activation [6
] suggesting that actin modification may play a role in such inhibition. Data presented here strongly suggests that changes in the actin cytoskeletal dynamics may indeed contribute to the loss of nuclear retinoid receptor function following T cell activation. Future studies should reveal the relationship between CFL1-mediated IS formation and modulation of retinoid receptor function.