PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E3-ligase MIR

Nagano, Koki; Bornhäuser, Beat; Warnasuriya, Gayathri D.; Entwistle, Alan; Cramer, Rainer; Lindholm, Dan; Naaby-Hansen, Søren

doi:10.1038/sj.emboj.7601059

Cited by 22 publications

(21 citation statements)

References 29 publications

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“…It should be noted that the staining of the nucleus, in addition to the filamentous structures, was also observed in the serum-starved cells, suggesting that the phosphorylated MLC 20 at Ser1/Ser2 seems to be present in the nucleus. This observation is similar to the previous reports that the phosphorylated MLC 20 was found in the nucleus (Matsumura et al, 1998;Nagano et al, 2006). After PDGF stimulation, the disassembly of the stress fibers was observed ( Figure 2A, j-l).…”

Section: Phosphorylation Of Mlc 20 At the Ser1/ser2 Sites Correlates supporting

confidence: 92%

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

Komatsu

Ikebe

2007

MBoC

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Phosphorylation of the regulatory light chain of myosin II (MLC 20 ) at the activation sites promotes both the motor activity and the filament formation of myosin II, thus playing an important role in various cell motile processes. In contrast, the physiological function of phosphorylation of MLC 20 at the inhibitory sites is unknown. Here we report for the first time the function of the inhibitory site phosphorylation in the cells. We successfully produced the antibodies specifically recognizing the phosphorylation sites of MLC 20 at Ser1, and the platelet-derived growth factor (PDGF)-induced change in the phosphorylation at the Ser1 was monitored. The phosphorylation of MLC 20 at the Ser1 significantly increased during the PDGF-induced actin cytoskeletal reorganization. PDGF disassembled the stress fibers, and this was attenuated with the expression of unphosphorylatable MLC 20 at the Ser1/Ser2 phosphorylation sites. The present results suggest that the down-regulation of myosin II activity achieved by the phosphorylation at the Ser1/Ser2 sites plays an important role in the normal reorganization of actomyosin filaments triggered by PDGF receptor stimulation. INTRODUCTIONCell migration plays a key role in both the physiological and the pathophysiological function of the cells including development, wound healing, immunity, and metastasis (Lauffenburger and Horwitz, 1996). Reorganization of actomyosin filaments is an essential process for these cell behaviors. It has been thought that myosin II plays a fundamental role in various types of cellular motility.In vitro biochemical studies have revealed that the function of smooth muscle and nonmuscle myosin II is regulated by the phosphorylation of MLC 20 (Sellers, 1991;Tan et al., 1992). A number of studies have shown that the phosphorylation of MLC 20 at Thr18 and Ser19 activates its motor activity and increases filament stability. On the other hand, it has been known that MLC 20 can be phosphorylated at other sites different from the activation sites. Originally, it was found that protein kinase C (PKC) phosphorylates Ser1/Ser2 and Thr9 of MLC 20 . This phosphorylation decreases the affinity of myosin II phosphorylated at the activation sites for actin and the affinity of MLC 20 for myosin light-chain kinase (MLCK; Nishikawa et al., 1984;Bengur et al., 1987;Ikebe et al., 1987;Ikebe and Reardon, 1990). In other words, the phosphorylation of MLC 20 at these sites inhibits rather than activates myosin II.It was reported that the phosphorylation of MLC 20 at the Thr9 inhibits myosin II motor activity and the phosphorylation of MLC 20 by MLCK (Nishikawa et al., 1984;Turbedsky et al., 1997); however, phosphorylation of the inhibitory sites in nonmuscle cells has only been observed on Ser1 and/or Ser2, but not on Thr9 in vivo (Kawamoto et al., 1989;Yamakita et al., 1994), and this is because the Ser1/Ser2 sites are resistant to dephosphorylation by myosin light chain phosphatases while phospho-Thr9 is readily dephosphorylated (Ikebe et al., 1999). Furthermore, it has b...

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Section: Phosphorylation Of Mlc 20 At the Ser1/ser2 Sites Correlates supporting

confidence: 92%

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

Komatsu

Ikebe

2007

MBoC

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“…Perhaps the most compelling mediator is the Akt substrate and 14-3-3-binding protein Kank, which inhibits RhoA activity and induces actin stress fiber disassembly in fibroblasts (4). Disassembly of actin stress fibers by insulin is reported to be mediated by the mitogen activated protein kinase p38 (58) or by phosphatidylinositol 5-phosphate (59), whereas PDGF actions are reported to require p41/42 activity (60) or an E3 ubiquitin ligase specific for myosin regulatory light chain (61). Hence, how Akt signaling induces disassembly of actin stress fibers and whether this involves multiple nonredundant mechanisms are unresolved questions.…”

Section: Discussionmentioning

confidence: 99%

The Sodium-Hydrogen Exchanger NHE1 Is an Akt Substrate Necessary for Actin Filament Reorganization by Growth Factors

Meima

Webb

Witkowska

et al. 2009

Journal of Biological Chemistry

102

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The kinase Akt mediates signals from growth factor receptors for increased cell proliferation, survival, and migration, which contribute to the positive effects of Akt in cancer progression. Substrates are generally inhibited when phosphorylated by Akt; however, we show phosphorylation of the plasma membrane sodium-hydrogen exchanger NHE1 by Akt increases exchanger activity (H ؉ efflux). Our data fulfill criteria for NHE1 being a bona fide Akt substrate, including direct phosphorylation in vitro, using mass spectrometry and Akt phospho-substrate antibodies to identify Ser 648 as the Akt phosphorylation site and loss of increased exchanger phosphorylation and activity by insulin and platelet-derived growth factor in fibroblasts expressing a mutant NHE1-S648A. How Akt induces actin cytoskeleton remodeling to promote cell migration and tumor cell metastasis is unclear, but disassembly of actin stress fibers by platelet-derived growth factor and insulin and increased proliferation in growth medium are inhibited in fibroblasts expressing NHE1-S648A. We predict that other functions shared by Akt and NHE1, including cell growth and survival, might be regulated by increased H ؉ efflux.The serine/threonine kinase Akt/protein kinase B functions as a convergence site of cues that signal through the lipid kinase phosphoinositide 3-kinase (PI 3-kinase), 4 including activated integrin receptors, growth factor receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors. Akt confers signal relay from these upstream regulators to increase cell proliferation, cell survival, protein synthesis, and glycolytic flux (1). These actions contribute to the positive effects of Akt in cancer progression, and PI 3-kinase/Akt signaling is commonly aberrant in human tumors (2). Akt also regulates reorganization of the actin cytoskeleton, including disassembly of bundled actin stress fibers and formation of actin filament-rich membrane protrusions (3, 4), which likely enhance the positive effects of some Akt isoforms on cell migration (5) and tumor cell metastasis (6). However, compared with most cell processes regulated by Akt, our understanding of how Akt regulates cytoskeleton dynamics is least understood. Akt generally phosphorylates substrates at an RXRXX(S/T) recognition motif (7), and in most instances Akt phosphorylation inhibits activity or function of the substrate. Akt promotes cell proliferation by phosphorylating and inhibiting the negative cell cycle regulators p21Cip1/WAF1 (8) and p27 Kip1 (9 -11) and Wee1 kinase (12). It promotes cell survival by phosphorylating and inhibiting the function of proapoptosis regulators, including Bad (13,14) and forkhead transcription factors (15,16), and it increases protein synthesis by phosphorylating and inhibiting the negative regulators of mTOR, tubersclerosis sclerosis protein 2 (17) and proline-rich Akt substrate of 40 kDa (18). Additionally, Akt increases metabolism in part by phosphorylating and inhibiting glycogen synthase 3 (19). However, there are a few exceptions w...

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“…Heterogeneous nuclear ribonucleoproteins (hnRNPs) are predominantly nuclear RNA-binding proteins that have been implicated in diverse molecular and cellular functions, including chromatin remodeling, transcription, splicing and translation (14,(25)(26)(27)(28). It acts as a docking platform to integrate signaling cascades by facilitating cross-talk between kinases and factors that mediate nucleic acid-directed processes (29).…”

Section: Identification Of Target Protein For Intracellular Antibody-mentioning

confidence: 99%

Loss-of-function screening by randomized intracellular antibodies: Identification of hnRNP-K as a potential target for metastasis

Inoue

Sawata

Taira

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a loss-of-function screening system on the basis of intracellular expression of single domain antibodies. We demonstrate its use in identification of potential targets of metastasis of human cancerous cells. Randomized intracellular antibodies were expressed in highly metastatic cells, and a derivative pool of cells with loss of migration phenotype in chemotaxis assay was isolated. Isolation of antibodies from cells with loss of migration phenotype and identification of their target proteins revealed the involvement of the heterogeneous nuclear ribonucleoprotein K (hnRNP-K), a multifunctional signaling protein, in metastasis. Furthermore, we found that the cytoplasmic accumulation of hnRNP-K is crucial for its role in metastasis. The results demonstrate (i) the advantages of our functional interference screening over the gene-knockouts and genesilencing, (ii) hnRNP-K as a potential target of metastasis, and (iii) a potential anti-metastasis peptide validated in in vitro cell migration assays.heterogeneous nuclear ribonucleoprotein K ͉ randomized antibodies ͉ functional screening ͉ cytoplasmic accumulation ͉ antimetastasis M etastasis is a complex phenomenon that involves an array of genetic changes and coordination of many positive and negative factors leading to motile cells that can penetrate into and travel through the bloodstream forming secondary tumors elsewhere in the body. Despite the advances in cancer therapies and the understanding that metastasis is a selective, nonrandom and inefficient process, it significantly contributes to morbidity and mortality of cancer patients and is a major challenge yet to be met in cancer therapeutics (1-4). Increased cell motility is one of the early and essential phenotype of metastatic cancers. Although it has been demonstrated to involve concerted reorganization of the actin cytoskeleton that regulates cell shape, and motility through spatiotemporal activation of Rho-family GTPases (5-9), the molecular mechanisms and its upstream regulators, the potential targets of metastasis, have not been sufficiently defined (10). Amongst a large variety of approaches that can be adopted to identify ''metastasis genes,'' we chose to employ a functional interference of proteins by expression of intracellular antibodies, also called as ''specific functional ablators'' and ''functional therapeutical molecules'' (11-13). We anticipated that the functional interference may serve as a milder and finer approach to reveal biological complexities and pathways that cannot be resolved by gene-knockouts and genesilencing strategies that challenge structural integrity of cells.We constructed a randomized intracellular antibody library (iAb-lib) on the basis of the single heavy-chain small antigenbinding fragments (VHH) of camel antibody. By employing iAb-lib against cell migration of highly malignant human fibrosarcoma, we obtained a derivative cell population in which cell migration was compromised. Cloning and characterization of antibodies from the derivative nonmigrating...

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PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E3-ligase MIR

Cited by 22 publications

References 29 publications

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

The Sodium-Hydrogen Exchanger NHE1 Is an Akt Substrate Necessary for Actin Filament Reorganization by Growth Factors

Loss-of-function screening by randomized intracellular antibodies: Identification of hnRNP-K as a potential target for metastasis

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