Increased α-actinin-1 destabilizes E-cadherin-based adhesions and associates with poor prognosis in basal-like breast cancer.

The controlled formation and stabilization of E-cadherin-based adhesions is vital for epithelial integrity. This requires co-operation between the E-cadherin-based adhesions and the associated actin cytoskeleton. In cancer, this co-operation often fails, predisposing cells to migration through molecular mechanisms that have only been partially characterized. Here, we demonstrate that the actin filament cross-linker α-actinin-1 is frequently increased in human breast cancer. In mammary epithelial cells, the increased α-actinin-1 levels promote cell migration and induce disorganized acini-like structures in Matrigel. This is accompanied by a major reorganization of the actin cytoskeleton and the associated E-cadherin-based adhesions. Increased expression of α-actinin-1 is particularly noted in basal-like breast cancer cell lines, and in breast cancer patients it associates with poor prognosis in basal-like subtypes. Downregulation of α-actinin-1 in E-cadherin expressing basal-like breast cancer cells demonstrate that α-actinin-1-assembled actin fibers destabilize E-cadherin-based adhesions. Taken together, these results indicate that increased α-actinin-1 expression destabilizes E-cadherin-based adhesions, which is likely to promote the migratory potential of breast cancer cells. Furthermore, our results identify α-actinin-1 as a candidate prognostic biomarker in basal-like breast cancer.

SUMOylation of AMPKα1 by PIAS4 specifically regulates mTORC1 signalling.

AMP-activated protein kinase (AMPK) inhibits several anabolic pathways such as fatty acid and protein synthesis, and identification of AMPK substrate specificity would be useful to understand its role in particular cellular processes and develop strategies to modulate AMPK activity in a substrate-specific manner. Here we show that SUMOylation of AMPKα1 attenuates AMPK activation specifically towards mTORC1 signalling. SUMOylation is also important for rapid inactivation of AMPK, to allow prompt restoration of mTORC1 signalling. PIAS4 and its SUMO E3 ligase activity are specifically required for the AMPKα1 SUMOylation and the inhibition of AMPKα1 activity towards mTORC1 signalling. The activity of a SUMOylation-deficient AMPKα1 mutant is higher than the wild type towards mTORC1 signalling when reconstituted in AMPKα-deficient cells. PIAS4 depletion reduced growth of breast cancer cells, specifically when combined with direct AMPK activator A769662, suggesting that inhibiting AMPKα1 SUMOylation can be explored to modulate AMPK activation and thereby suppress cancer cell growth.

Actin stress fibre subtypes in mesenchymal-migrating cells.

Mesenchymal cell migration is important for embryogenesis and tissue regeneration. In addition, it has been implicated in pathological conditions such as the dissemination of cancer cells. A characteristic of mesenchymal-migrating cells is the presence of actin stress fibres, which are thought to mediate myosin II-based contractility in close cooperation with associated focal adhesions. Myosin II-based contractility regulates various cellular activities, which occur in a spatial and temporal manner to achieve directional cell migration. These myosin II-based activities involve the maturation of integrin-based adhesions, generation of traction forces, establishment of the front-to-back polarity axis, retraction of the trailing edge, extracellular matrix remodelling and mechanotransduction. Growing evidence suggests that actin stress fibre subtypes, namely dorsal stress fibres, transverse arcs and ventral stress fibres, could provide this spatial and temporal myosin II-based activity. Consistent with their functional differences, recent studies have demonstrated that the molecular composition of actin stress fibre subtypes differ significantly. This present review focuses on the current view of the molecular composition of actin stress fibre subtypes and how these fibre subtypes regulate mesenchymal cell migration.

Assembly of non-contractile dorsal stress fibers requires α-actinin-1 and Rac1 in migrating and spreading cells.

Cell migration and spreading is driven by actin polymerization and actin stress fibers. Actin stress fibers are considered to contain α-actinin crosslinkers and nonmuscle myosin II motors. Although several actin stress fiber subtypes have been identified in migrating and spreading cells, the degree of molecular diversity of their composition and the signaling pathways regulating fiber subtypes remain largely uncharacterized. In the present study we identify that dorsal stress fiber assembly requires α-actinin-1. Loss of dorsal stress fibers in α-actinin-1-depleted cells results in defective maturation of leading edge focal adhesions. This is accompanied by a delay in early cell spreading and slower cell migration without noticeable alterations in myosin light chain phosphorylation. In agreement with the unaltered myosin II activity, dorsal stress fiber trunks lack myosin II and are resistant to myosin II ATPase inhibition. Furthermore, the non-contractility of dorsal stress fibers is supported by the finding that Rac1 induces dorsal stress fiber assembly whereas contractile ventral stress fibers are induced by RhoA. Loss of dorsal stress fibers either by depleting α-actinin-1 or Rac1 results in a ß-actin accumulation at the leading edge in migrating and spreading cells. These findings molecularly specify dorsal stress fibers from other actin stress fiber subtypes. Furthermore, we propose that non-contractile dorsal stress fibers promote cell migration and early cell spreading through Rac1-induced actin polymerization.

An association between NUAK2 and MRIP reveals a novel mechanism for regulation of actin stress fibers.

Actin stress fiber assembly and contractility in nonmuscle motile cells requires phosphorylation of myosin regulatory light chain (MLC). Dephosphorylation and disassembly are mediated by MLC phosphatase, which is targeted to actin fibers by the association of its regulatory subunit MYPT1 with myosin phosphatase Rho-interacting protein (MRIP). In the present study, we identify the kinase NUAK2 as a second protein targeted by MRIP to actin fibers. Association of NUAK2 with MRIP increases MLC phosphorylation and promotes formation of stress fibers. This activity does not require the kinase activity of NUAK2 but is dependent on both MRIP and MYPT1, indicating that the NUAK2-MRIP association inhibits fiber disassembly and MYPT1-mediated MLC dephosphorylation. NUAK2 levels are strongly induced by stimuli increasing actomyosin fiber formation, and NUAK2 is required for fiber maintenance in exponentially growing cells, implicating NUAK2 in a positive-feedback loop regulating actin stress fibers independently of the MLC kinase Rho-associated protein kinase (ROCK). The identified MRIP-NUAK2 association reveals a novel mechanism for the maintenance of actin stress fibers through counteracting MYPT1 and, together with recent results, implicates the NUAK proteins as important regulators of the MLC phosphatase acting in both a kinase-dependent and kinase-independent manner.

Cyclin G-associated kinase promotes microtubule outgrowth from chromosomes during spindle assembly.

During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation. Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the mitotic spindle. Here, we have performed a large scale RNAi screen and identify cyclin G-associated kinase (GAK) as a novel regulator of microtubule generation at kinetochores/chromatin. This function of GAK requires its C-terminal J-domain, which is essential for clathrin recycling from endocytic vesicles. Consistently, cells lacking GAK show strongly reduced levels of clathrin on the mitotic spindle, and reduction of clathrin levels also inhibits microtubule generation at kinetochores/chromosomes. Finally, we present evidence that association of clathrin with the spindle is promoted by a signal coming from the chromosomes. These results identify a role for GAK and clathrin in microtubule outgrowth from kinetochores/chromosomes and suggest that GAK acts through clathrin to control microtubule outgrowth around chromosomes.

Integrated network analysis platform for protein-protein interactions.

There is an increasing demand for network analysis of protein-protein interactions (PPIs). We introduce a web-based protein interaction network analysis platform (PINA), which integrates PPI data from six databases and provides network construction, filtering, analysis and visualization tools. We demonstrated the advantages of PINA by analyzing two human PPI networks; our results suggested a link between LKB1 and TGFbeta signaling, and revealed possible competitive interactors of p53 and c-Jun.

Lkb1 is required for TGFbeta-mediated myofibroblast differentiation.

Inactivating mutations of the tumor-suppressor kinase gene LKB1 underlie Peutz-Jeghers syndrome (PJS), which is characterized by gastrointestinal hamartomatous polyps with a prominent smooth-muscle and stromal component. Recently, it was noted that PJS-type polyps develop in mice in which Lkb1 deletion is restricted to SM22-expressing mesenchymal cells. Here, we investigated the stromal functions of Lkb1, which possibly underlie tumor suppression. Ablation of Lkb1 in primary mouse embryo fibroblasts (MEFs) leads to attenuated Smad activation and TGFbeta-dependent transcription. Also, myofibroblast differentiation of Lkb1(-/-) MEFs is defective, resulting in a markedly decreased formation of alpha-smooth muscle actin (SMA)-positive stress fibers and reduced contractility. The myofibroblast differentiation defect was not associated with altered serum response factor (SRF) activity and was rescued by exogenous TGFbeta, indicating that inactivation of Lkb1 leads to defects in myofibroblast differentiation through attenuated TGFbeta signaling. These results suggest that tumorigenesis by Lkb1-deficient SM22-positive cells involves defective myogenic differentiation.

LKB1 in endothelial cells is required for angiogenesis and TGFbeta-mediated vascular smooth muscle cell recruitment.

Inactivation of the tumor suppressor kinase Lkb1 in mice leads to vascular defects and midgestational lethality at embryonic day 9-11 (E9-E11). Here, we have used conditional targeting to investigate the defects underlying the Lkb1(-/-) phenotype. Endothelium-restricted deletion of Lkb1 led to embryonic death at E12.5 with a loss of vascular smooth muscle cells (vSMCs) and vascular disruption. Transforming growth factor beta (TGFbeta) pathway activity was reduced in Lkb1-deficient endothelial cells (ECs), and TGFbeta signaling from Lkb1(-/-) ECs to adjacent mesenchyme was defective, noted as reduced SMAD2 phosphorylation. The addition of TGFbeta to mutant yolk sac explants rescued the loss of vSMCs, as evidenced by smooth muscle alpha actin (SMA) expression. These results reveal an essential function for endothelial Lkb1 in TGFbeta-mediated vSMC recruitment during angiogenesis.

The LKB1 tumor suppressor kinase in human disease.

Inactivating germline mutations in the LKB1 gene underlie Peutz-Jeghers syndrome characterized by hamartomatous polyps and an elevated risk for cancer. Recent studies suggest the involvement of LKB1 also in more common human disorders including diabetes and in a significant fraction of lung adenocarcinomas. These observations have increased the interest towards signaling pathways of this tumor suppressor kinase. The recent breakthroughs in understanding the molecular functions of the LKB1 indicate its contribution as a regulator of cell polarity, energy metabolism and cell proliferation. Here we review how the substrates and cellular functions of LKB1 may be linked to Peutz-Jeghers syndrome and other diseases, and discuss how some of the molecular changes associated with altered LKB1 signaling might be used in therapeutic approaches.

The PDZ-LIM protein RIL modulates actin stress fiber turnover and enhances the association of alpha-actinin with F-actin.

ALP, CLP-36 and RIL form the ALP subfamily of PDZ-LIM proteins. ALP has been implicated in sarcomere function in muscle cells in association with alpha-actinin. The closely related CLP-36 is predominantly expressed in nonmuscle cells, where it localizes to actin stress fibers also in association with alpha-actinin. Here we have studied the expression and functions of RIL originally identified as a gene downregulated in H-ras-transformed cells. RIL was mostly expressed in nonmuscle epithelial cells with a pattern distinct from that of CLP-36. RIL protein was found to localize to actin stress fibers in nonmuscle cells similarly to CLP-36. However, RIL expression led to partially abnormal actin filaments showing thick irregular stress fibers not seen with CLP-36. Furthermore, live cell imaging demonstrated altered stress fiber dynamics with rapid formation of new fibers and frequent collapse of thick irregular fibers in EGFP-RIL-expressing cells. These effects may be mediated through the association of RIL with alpha-actinin, as RIL was found to associate with alpha-actinin via its PDZ domain, and RIL enhanced the ability of alpha-actinin to cosediment with actin filaments. These results implicate the RIL PDZ-LIM protein as a regulator of actin stress fiber turnover.

Clik1: a novel kinase targeted to actin stress fibers by the CLP-36 PDZ-LIM protein.

In this report we have characterized a novel, ubiquitously expressed kinase, Clik1, that is predominantly nuclear and undergoes autophosphorylation. Yeast two-hybrid analysis indicated a highly specific association between Clik1 and CLP-36, which was identified in 36 out of 37 Clik1-interacting clones. CLP-36 is a PDZ-LIM protein that localizes to actin stress fibers in nonmuscle cells and associates with alpha-actinin via its PDZ-domain. The association of CLP-36 with Clik1, in turn, is mediated by the C-terminal part of CLP-36 containing the LIM domain, and association was not noted with the closely related ALP PDZ-LIM protein. Interestingly, the association with CLP-36 led to relocalization of the otherwise nuclear Clik1 kinase to actin stress fibers, where it disrupted the periodic staining pattern of CLP-36. Taken together these results establish the CLP-36 PDZ-LIM protein as an adapter, recruiting the Clik1 kinase to actin stress fibers in nonmuscle cells, and suggest that Clik1 represents a novel regulator of actin stress fibers.