Cai Huang

MAP kinases and cell migration.

Recent studies have demonstrated that mitogen-activated protein kinases (MAPKs), including Jun N-terminus kinase (JNK), p38 and Erk, play crucial roles in cell migration. JNK, for example, regulates cell migration by phosphorylating paxillin, DCX, Jun and microtubule-associated proteins. Studies of p38 show that this MAPK modulates migration by phosphorylating MAPK-activated protein kinase 2/3 (MAPKAP 2/3), which appears to be important for directionality of migration. Erk governs cell movement by phosphorylating myosin light chain kinase (MLCK), calpain or FAK. Thus, the different kinases in the MAPK family all seem able to regulate cell migration but by distinct mechanisms.

JNK phosphorylates paxillin and regulates cell migration

The c-Jun amino-terminal kinase (JNK) is generally thought to be involved in inflammation, proliferation and apoptosis. Accordingly, its substrates are transcription factors and anti-apoptotic proteins. However, JNK has also been shown to be required for Drosophila dorsal closure, and MAP kinase/ERK kinase kinase 1, an upstream kinase in the JNK pathway, has been shown to be essential for cell migration. Both results imply that JNK is important in cell migration. Here we show that JNK1 is required for the rapid movement of both fish keratocytes and rat bladder tumour epithelial cells (NBT-II). Moreover, JNK1 phosphorylates serine 178 on paxillin, a focal adhesion adaptor, both in vitro and in intact cells. NBT-II cells expressing the Ser 178 → Ala mutant of paxillin (PaxS178A) formed focal adhesions and exhibited the limited movement associated with such contacts in both single-cell-migration and wound-healing assays. In contrast, cells expressing wild-type paxillin moved rapidly and retained close contacts as the predominant adhesion. Expression of PaxS178A also inhibited the migration of two other cell lines. Thus, phosphorylation of paxillin by JNK seems essential for maintaining the labile adhesions required for rapid cell migration.

Talin phosphorylation by Cdk5 regulates Smurf1-mediated talin head ubiquitylation and cell migration.

Cell migration is a dynamic process that requires temporal and spatial regulation of integrin activation and focal adhesion assembly/disassembly. Talin, an actin and β-integrin tail-binding protein, is essential for integrin activation and focal adhesion formation. Calpain-mediated cleavage of talin has a key role in focal adhesion turnover; however, the talin head domain, one of the two cleavage products, stimulates integrin activation, localizes to focal adhesions and maintains cell edge protrusions, suggesting that other steps, downstream of talin proteolysis, are required for focal adhesion disassembly. Here we show that talin head binds Smurf1, an E3 ubiquitin ligase involved in cell polarity and migration, more tightly than full-length talin does and that this interaction leads to talin head ubiquitylation and degradation. We found that talin head is a substrate for Cdk5, a cyclin-dependent protein kinase that is essential for cell migration, synaptic transmission and cancer metastasis. Cdk5 phosphorylated talin head at Ser 425, inhibiting its binding to Smurf1, thus preventing talin head ubiquitylation and degradation. Expression of the mutant talS425A, which resists Cdk5 phosphorylation thereby increasing its susceptibility to Smurf1-mediated ubiqitylation, resulted in extensive focal adhesion turnover and inhibited cell migration. Thus, talin head produced by calpain-induced cleavage of talin is degraded through Smurf1-mediated ubiquitylation; moreover, phosphorylation by Cdk5 regulates the binding of Smurf1 to talin head, controlling talin head turnover, adhesion stability and ultimately, cell migration.

Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC-12 cells

Cell adhesions play an important role in neurite extension. Paxillin, a focal adhesion adaptor protein involved in focal adhesion dynamics, has been demonstrated to be required for neurite outgrowth. However, the molecular mechanism by which paxillin regulates neurite outgrowth is unknown. Here, we show that paxillin is phosphorylated by p38MAPK in vitro and in nerve growth factor (NGF)–induced PC-12 cells. Ser 85 (Ser 83 for endogenous paxillin) is identified as one of major phosphorylation sites by phosphopeptide mapping and mass spectrometry. Moreover, expression of the Ser 85 → Ala mutant of paxillin (paxS85A) significantly inhibits NGF-induced neurite extension of PC-12 cells, whereas expression of wild-type (wt) paxillin does not influence neurite outgrowth. Further experiments indicate that cells expressing paxS85A exhibit small, clustered focal adhesions which are not normally seen in cells expressing wt paxillin. Although wt paxillin and paxS85A have the same ability to bind vinculin and focal adhesion kinase, wt paxillin more efficiently associates with Pyk2 than paxS85A. Thus, phosphorylation of paxillin is involved in NGF-induced neurite extension of PC-12 cells, probably through regulating focal adhesion organization.

Proteolysis of platelet cortactin by calpain.

Cortactin, a substrate of pp60c- src and a potent filamentous actin binding and cross-linking protein, is abundant in circulating platelets. After stimulation of platelet aggregation with collagen, cortactin undergoes a dramatic increase in tyrosine phosphorylation followed by a rapid degradation. The cleavage of platelet cortactin was detected in lysates prepared using either Triton-containing buffer or SDS-sample buffer. However, the degradation of cortactin was not observed in platelets derived from a Glanzmann’s patient, who lacked functional integrin αIIbβ3 (GPIIb-IIIa). In addition, the proteolysis of cortactin was abolished by treating platelets before but not after collagen stimulation with EGTA or calpeptin. Furthermore, recombinant cortactin was digested by μ-calpain in vitro in a dose-dependent manner, indicating that cortactin is a substrate for calpain. We also observed that the calpain-mediated digestion in vitro is dependent on the presence of a sequence containing a proline-rich region and multiple tyrosine residues that are phosphorylated by pp60c- src . Tyrosine phosphorylation by pp60c- src up-regulates the activity of calpain toward cortactin. Our data suggest that the calpain-mediated proteolysis of tyrosine-phosphorylated cortactin may provide a mechanism to remodel irreversibly the cytoskeleton in response to platelet agonists.

Src is required for cell migration and shape changes induced by fibroblast growth factor 1

Fibroblast growth factor 1 (FGF-1) is a potent chemotactic factor and induces tyrosine phosphorylation of a cortical actin-associated protein (cortactin). The tyrosine phosphorylation of cortactin induced by FGF-1 requires the tyrosine residues 421, 482 and 466, which are targeted by the protein tyrosine kinase Src in vitro. Furthermore, FGF-1 is unable to induce tyrosine phosphorylation of cortactin within the cells derived from Src knockout mice (Src−/−), indicating that Src is required for the tyrosine phosphorylation of cortactin induced by FGF-1. Although Src−/− cells are able to undergo rapid proliferation, they are impaired to respond to FGF-1 for the shape change and cell migration. Morphological analysis further reveals that FGF-1 fails to induce the formation of polarized lamellipodia and the translocation of cortactin into the leading edge of Src−/− cells. Consistent with the mitogenic response to FGF-1, the lack of Src does not affect the tyrosine phosphorylation of Snt (or Frs2), a FGF-1 early signaling protein that links to Ras. Therefore, our data support the notion that Src and cortactin participate in a FGF signal pathway for cell migration and shape change rather than mitogenesis.

A role for JNK-paxillin signaling in cell migration

Recently, we and others demonstrated that JNK is essential for cell migration in a number of cell types. We also showed that JNK phosphorylates serine 178 on paxillin, a focal adhesion adaptor, both in vitro and in vivo. Moreover, phosphorylation of Ser 178 on paxillin is essential for cell migration and involved in modulating cell adhesions. A model is proposed to depict the role of JNK-paxillin signaling in cell migration.

Roles of E3 ubiquitin ligases in cell adhesion and migration.

Recent studies have demonstrated that a number of E3 ubiquitin ligases, including Cbl, Smurf1, Smurf2, HDM2, BCA2, SCFβ-TRCP and XRNF185, play important roles in cell adhesion and migration. Cbl negatively regulates cell adhesion via α integrin and Rap1, and inhibits actin polymerization by ubiquitinating mDab1 and WAVE2. Smurf1 regulates cell migration through ubiquitination of RhoA, talin head domain and hPEM2, while Smurf2 ubiquitinates Smurf1, TGF-β type I receptor and RaplB to modulate cell migration and adhesion. HDM2 negatively regulates cell migration by targeting NFAT (a transcription factor) for ubiquitination and degradation, while SCFβ-TRCP ubiquitinates Snail (a transcriptional repressor of E-cadherin) to inhibit cell migration. TRIM32 promotes cell migration through ubiquitination of Abl interactor 2 (Abi2), a tumor suppressor. RNF5 and XRNF185 modulate cell migration by ubiquitinating paxillin. Thus, theses E3 ubiquitin ligases regulate cell adhesion and (or) migration through ubiquitination of their specific substrates.

Talin1 phosphorylation activates β1 integrins: a novel mechanism to promote prostate cancer bone metastasis

Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that β1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how β1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of β1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in β1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1S425A, in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of β1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1S425D led to increased β1 integrin activation and generated biologic effects opposite to talin1S425A expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1S425A, in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1S425D restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent β1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to β1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells.

Ubiquitylation of phosphatidylinositol 4-phosphate 5-kinase type I γ by HECTD1 regulates focal adhesion dynamics and cell migration.

Summary Phosphatidylinositol 4-phosphate 5-kinase type I &ggr; (PIPKI&ggr;90) binds talin and localizes at focal adhesions (FAs). Phosphatidylinositol (4,5)-bisphosphate (PIP2) generated by PIPKI&ggr;90 is essential for FA formation and cell migration. On the other hand, PIPKI&ggr;90 and the &bgr;-integrin tail compete for overlapping binding sites on talin. Enhanced PIPKI&ggr;90-talin interaction suppresses talin binding to the &bgr;-integrin. It is unknown how PIPKI&ggr;90 is removed from the PIPKI&ggr;90–talin complex after on-site PIP2 production during cell migration. Here we show that PIPKI&ggr;90 is a substrate for HECTD1, an E3 ubiquitin ligase regulating cell migration. HECTD1 ubiquitinated PIPKI&ggr;90 at lysine 97 and resulted in PIPKI&ggr;90 degradation. Expression of the mutant PIPKI&ggr;90K97R enhanced PIP2 and PIP3 production, inhibited FA assembly and disassembly and inhibited cancer cell migration, invasion and metastasis. Interestingly, mutation at tryptophan 647 abolished the inhibition of PIPKI&ggr;90K97R on FA dynamics and partially rescued cancer cell migration and invasion. Thus, cycling PIPKI&ggr;90 ubiquitylation by HECTD1 and consequent degradation remove PIPKI&ggr;90 from talin after on-site PIP2 production, providing an essential regulatory mechanism for FA dynamics and cell migration.