Woo Keun Song

The 31-kDa Caspase-generated Cleavage Product of p130cas Functions as a Transcriptional Repressor of E2A in Apoptotic Cells

In response to integrin receptor binding to the extracellular matrix, the multidomain docking protein p130cas (Crk-associated substrate) activates various signaling cascades modulating such cellular processes as proliferation, migration, and apoptosis. During apoptosis, caspase-mediated cleavage of p130cas generated a C-terminal 31-kDa fragment (31-kDa) and promoted morphological changes characteristic of apoptosis, including loss of focal adhesions, cell rounding, and nuclear condensation and fragmentation. By contrast, a p130cas D748E mutant, which was unable to produce 31-kDa, attenuated the disassembly of focal adhesions at the bottom of the cell. 31-kDa contains a helix-loop-helix (HLH) domain that shows greater sequence homology with Id proteins than with basic HLH proteins, which enabled heterodimerization with E2A. Once coupled to E2A, 31-kDa was translocated to the cell nucleus, where it inhibited E2A-mediated p21Waf1/Cip1 transcription. Moreover, overexpression of 31-kDa led to cell death that could be inhibited by treatment with the caspase inhibitor ZVAD-fluoromethyl ketone or by ectopic expression of E2A or p21Waf1/Cip1. These data suggest that during etoposide-induced apoptosis, 31-kDa promotes caspase-mediated cell death by inhibiting E2A-mediated activation of p21Waf1/Cip1 transcription.

Phosphorylation of Ser 21 in Fyn regulates its kinase activity, focal adhesion targeting, and is required for cell migration

The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin‐mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase‐linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell‐extracellular matrix interactions. J. Cell. Physiol. 226: 236–247, 2010.

Interaction of SPIN90 with the Arp2/3 complex mediates lamellipodia and actin comet tail formation.

The appropriate regulation of the actin cytoskeleton is essential for cell movement, changes in cell shape, and formation of membrane protrusions like lamellipodia and filopodia. Moreover, several regulatory proteins affecting actin dynamics have been identified in the motile regions of cells. Here, we provide evidence for the involvement of SPIN90 in the regulation of actin cytoskeleton and actin comet tail formation. SPIN90 was distributed throughout the cytoplasm in COS-7 cells, but exposing the cells to platelet-derived growth factor (PDGF) caused a redistribution of SPIN90 to the cell cortex and the formation of lamellipodia (or membrane ruffles), both of which were dramatically inhibited in SPIN90-knockdown cells. In addition, the binding of the C terminus of SPIN90 with both the Arp2/3 complex (actin-related proteins Arp 2 and Arp 3) and G-actin activates the former, leading to actin polymerization in vitro. And when coexpressed with phosphatidylinositol 4-phosphate 5 kinase, SPIN90 was observed within actin comet tails. Taken these findings suggest that SPIN90 participates in reorganization of the actin cytoskeleton and in actin-based cell motility.

Sphingosine 1-phosphate activates Erk-1/-2 by transactivating epidermal growth factor receptor in rat-2 cells.

We investigated a signaling pathway leading to activation of extracellular signal‐regulated protein kinase (Erk) 1 and 2 in Rat‐2 cells stimulated with sphingosine 1‐phosphate (S1P). S1P treatment transiently activated Erk‐1/‐2 in a dose‐dependent manner, and its activation was blocked by pertussis toxin, expression of RasN17, or inhibition of Raf or MEK‐1/‐2. S1P‐induced activation of Erk‐1/‐2 was also suppressed by the inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with the specific inhibitor AG1478, suggesting that activation of EGF receptor tyrosine kinase was involved in the signaling pathway. S1P‐induced Erk‐1/‐2 activation was enhanced up to 2‐fold by inhibiting protein kinase C (PKC) with GF109203X, and PKC inhibition in the absence of S1P treatment also activated Erk‐1/‐2. The stimulatory effects of Erk‐1/‐2 activation by PKC inhibition was blocked by treating cells with AG1478, suggesting the involvement of PKC in the regulation of EGF receptor tyrosine kinase activation that leads to Erk‐1/‐2 activation. Together, these results suggest that S1P activates the EGF receptor through a PKC‐dependent pathway that links Ras signaling to the activation of Erk‐1/‐2 in Rat‐2 cells.

Caspase-dependent cleavage of tensin induces disruption of actin cytoskeleton during apoptosis.

Members of both calpain and caspase protease families can degrade several components of focal adhesions, leading to disassembly of these complexes. In this report, we investigated the disappearance of tensin from cell adhesion sites of chicken embryonic fibroblast cells (CEFs) exposed to etoposide and demonstrated that loss of tensin from cell adhesions during etoposide-induced apoptosis may be due to degradation of tensin by caspase-3. Tensin cleavage by caspase-3 at the sequence DYPD(1226)G separates the amino-terminal region containing the actin binding domain and the carboxyl-terminal region containing the SH2 domain. The resultant carboxyl-terminal fragment of tensin is unable to bind phosphoinositide 3-kinase (PI3-kinase) transducing cell survival signaling. We also demonstrated that overexpression of the amino-terminal tensin fragment induced disruption of actin cytoskeleton in chicken embryonic fibroblasts. Therefore, caspase-mediated cleavage of tensin contributes to the disruption of actin organization and interrupts ECM-mediated survival signals through phosphatidylinositol 3-kinase.

Regulation of SPIN90 Phosphorylation and Interaction with Nck by ERK and Cell Adhesion

SPIN90 is a widely expressed Nck-binding protein that contains one Src homology 3 (SH3) domain, three Pro-rich motifs, and a serine/threonine-rich region, and is known to participate in sarcomere assembly during cardiac myocyte differentiation. We used in vitro binding assays and yeast two-hybrid screening analysis to identify Nck, βPIX, Wiscott-Aldrich syndrome protein (WASP), and ERK1 as SPIN90-binding proteins. It appears that βPIX, WASP, and SPIN90 form a complex that interacts with Nck in a manner dependent upon cell adhesion to extracellular matrix. The βPIX·WASP·SPIN90·Nck interaction was abolished in suspended and cytochalasin D-treated cells, but was recovered when cells were replated on fibronectin-coated dishes. The SPIN90·βPIX·WASP complex was stable, even in suspended cells, suggesting SPIN90 serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions. In addition, we found that overexpression of the SPIN90 SH3 domain or Pro-rich region, respectively, abolished SPIN90·Nck and SPIN90·βPIX interactions, resulting in detachment of cells from extracellular matrix. SPIN90 was phosphorylated by ERK1, which was, itself, activated by cell adhesion and platelet-derived growth factor. Such phosphorylation of SPIN90 likely promotes the interaction of the SPIN90·βPIX·WASP complex and Nck. It thus appears that the interaction of the βPIX·WASP·SPIN90 complex with Nck is crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERK activation.

SPIN90/WISH interacts with PSD‐95 and regulates dendritic spinogenesis via an N‐WASP‐independent mechanism

SPIN90/WISH (SH3 protein interacting with Nck, 90 kDa/Wiskott–Aldrich syndrome protein (WASP) interacting SH3 protein) regulates actin polymerization through its interaction with various actin‐regulating proteins. It is highly expressed in the brain, but its role in the nervous system is largely unknown. We report that it is expressed in dendritic spines where it associates with PSD‐95. Its overexpression increased the number and length of dendritic filopodia/spines via an N‐WASP‐independent mechanism, and knock down of its expression with small interfering RNA reduced dendritic spine density. The increase in spinogenesis is accompanied by an increase in synaptogenesis in contacting presynaptic neurons. Interestingly, PSD‐95‐induced dendritic spinogenesis was completely abolished by knock down of SPIN90/WISH. Finally, in response to chemically induced long‐term potentiation, SPIN90/WISH associated with PSD‐95 and was redistributed to dendritic spines. Our results suggest that SPIN90/WISH associates with PSD‐95, and so becomes localized to dendritic spines where it modulates actin dynamics to control dendritic spinogenesis. They also raise the possibility that SPIN90/WISH is a downstream effector of PSD‐95‐dependent synaptic remodeling.

Interaction of SPIN90 with Dynamin I and Its Participation in Synaptic Vesicle Endocytosis

SH3 protein interacting with Nck, 90 kDa (SPIN90) is an Nck-binding protein that contains one Src homology 3 (SH3) domain, three proline-rich domains (PRDs), a serine/threonine-rich region, and a hydrophobic C-terminal region. Previously, we have shown that SPIN90 plays roles in the sarcomere assembly in cardiac muscles and in the formation of focal contacts in HeLa cells. Besides in heart, SPIN90 is also highly expressed in the brain, but its role in the neuronal system is completely unknown. Here, we found that SPIN90 is expressed in the presynaptic compartment in which it binds dynamin I, a key component of the endocytic machinery, and that it participates in synaptic vesicle endocytosis. Pull-down analysis and coimmunoprecipitation proved the associations of SPIN90 with dynamin I through SH3-PRD interaction. Overexpression of SPIN90 or knocking down SPIN90 by small interfering RNA impaired synaptic vesicle endocytosis. We further confirmed by the rescue experiments that the endocytic defects by SPIN90 expression come from its interaction with dynamin I. Exocytosis kinetics was not affected by SPIN90 expression. Together, our findings suggest that SPIN90 could modulate the interactions of dynamin I with other endocytic proteins that cooperate in the coated vesicle formation, thus regulating synaptic vesicle endocytosis.

Swiprosin-1 Is a Novel Actin Bundling Protein That Regulates Cell Spreading and Migration

Protein functions are often revealed by their localization to specialized cellular sites. Recent reports demonstrated that swiprosin-1 is found together with actin and actin-binding proteins in the cytoskeleton fraction of human mast cells and NK-like cells. However, direct evidence of whether swiprosin-1 regulates actin dynamics is currently lacking. We found that swiprosin-1 localizes to microvilli-like membrane protrusions and lamellipodia and exhibits actin-binding activity. Overexpression of swiprosin-1 enhanced lamellipodia formation and cell spreading. In contrast, swiprosin-1 knockdown showed reduced cell spreading and migration. Swiprosin-1 induced actin bundling in the presence of Ca2+, and deletion of the EF-hand motifs partially reduced bundling activity. Swiprosin-1 dimerized in the presence of Ca2+ via its coiled-coil domain, and a lysine (Lys)-rich region in the coiled-coil domain was essential for regulation of actin bundling. Consistent with these observations, mutations of the EF-hand motifs and coiled-coil region significantly reduced cell spreading and lamellipodia formation. We provide new evidence of how swiprosin-1 influences cytoskeleton reorganization and cell spreading.

Interaction of SPIN90 with syndapin is implicated in clathrin-mediated endocytic pathway in fibroblasts

SPIN90, a 90‐kDa Nck‐interacting protein with a SH3 domain, plays a role in sarcomere formation and myofibril assembly, and its phosphorylation is modulated by cell adhesion and Erk activation. Here we demonstrate that SPIN90 participates in receptor‐mediated endocytic pathway in fibroblasts. We identified syndapin (synaptic dynamin‐binding protein) as a SPIN90 interacting protein using yeast two‐hybrid screening. SPIN90 directly binds the SH3 domain of syndapin via its proline rich domain in vitro and in vivo and also associates with clathrin. Over‐expression of SPIN90‐full length in COS‐7 cells inhibited transferrin uptake, a marker of endocytosis. Interestingly, SPIN90‐PRD, a syndapin‐binding domain, significantly inhibited endocytosis, and the inhibition was reversed by co‐expression of syndapin. Depleting SPIN90 through antibody microinjection or Knocking it down using siRNAs also significantly inhibited transferrin internalization. Moreover, early endosomal marker proteins (EEA1 and Rab5) appeared to closely associate or partially co‐localize with SPIN90 in endosomes and an internalized FITC‐dextran and Texas Red‐EGF were found on the endosomes in association with SPIN90. Time‐lapse video showed that GFP‐SPIN90 travels with moving vesicles within living cells. Taken together, these findings suggest that SPIN90 is implicated in receptor‐mediated endocytic pathway in fibroblasts.