The Carboxy Terminus of AFAP-110 Modulates Direct Interactions with Actin Filaments and Regulates Its Ability to Alter Actin Filament Integrity and Induce Lamellipodia Formation

Qian, Yong; Baisden, Joseph M.; Zot, Henry G.; Winkle, W. B. Van; Flynn, Daniel C.

doi:10.1006/excr.1999.4795

Cited by 47 publications

(101 citation statements)

References 33 publications

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“…The full-length human AFAP (hAFAP) coding sequence was subcloned into the pCMV1 vector by replacing the resident KpnI-XbaI fragment of the pCMV-AFAP construct (21). The pEGFP-C3 eukaryotic expression vector (Clontech) was used to express cAFAP and its mutants fused to green fluorescent protein (GFP) by shuttling cAFAP, AFAP71A, AFAP77A, and AFAP5Y sequences from pCMV vectors to pEGFP-C3 vector, respectively, using EcoRI subcloning sites (22). Small interfering RNA (siRNA) was designed according to the nucleotides 311-331 of the human AFAP sequence (GenBank TM access number AF188700).…”

Section: Methodsmentioning

confidence: 99%

“…To determine whether AFAP can directly activate c-Src, an in vitro reconstitution assay was performed (23). Briefly, 20 ng of purified and C-terminal Src kinase-inactivated c-Src (a gift from Dr. X. Huang, Cornell University) in Src kinase buffer (30 mM HEPES, pH 7.4, 5 mM MgCl 2 , 5 mM MnCl 2 , and 10 M ATP) was incubated with 2 g of Src substrate peptide (24) and 10 Ci of [␥-32 P]ATP with purified recombinant cAFAP or AFAP71A (22) in a total 20-l reaction volume at 30°C for 15 min. The reaction was terminated by adding Laemmli sample buffer.…”

Section: Methodsmentioning

confidence: 99%

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Conversion of Mechanical Force into Biochemical Signaling

Han

Bai

Lodyga

et al. 2004

Journal of Biological Chemistry

144

132

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Physical forces play important roles in regulating cell proliferation, differentiation, and death by activating intracellular signal transduction pathways. How cells sense mechanical stimulation, however, is largely unknown. Most studies focus on cellular membrane proteins such as ion channels, integrins, and receptors for growth factors as mechanosensory units. Here we show that mechanical stretch-induced c-Src protein tyrosine kinase activation is mediated through the actin filament-associated protein (AFAP). Distributed along the actin filaments, AFAP can directly active c-Src through binding to its Src homology 3 and/or 2 domains. Mutations at these specific binding sites on AFAP blocked mechanical stretch-induced c-Src activation. Therefore, mechanical force can be transmitted along the cytoskeleton, and interaction between cytoskeletal associated proteins and enzymes related to signal transduction may convert physical forces into biochemical reactions. Cytoskeleton deformation-induced proteinprotein interaction via specific binding sites may represent a novel intracellular mechanism for cells to sense mechanical stimulation.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Conversion of Mechanical Force into Biochemical Signaling

Han

Bai

Lodyga

et al. 2004

Journal of Biological Chemistry

144

132

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“…The expression of AFAP-110 Dlzip results in a cell phenotype which resembles Src-transformed cells, while overexpression of wild-type AFAP-110 has no e ect on actin structures (Qian et al, 1998(Qian et al, , 2000. Cells expressing AFAP-110 Dlzip consistently displayed actin-rich rosettes in place of actin ®laments, as well as actin-rich lamellipodia.…”

Section: Deletion Of the Leucine Zipper Motif Enables Afap-110 To Altmentioning

confidence: 99%

“…AFAP-110 contains several putative protein binding domains, including SH2 and SH3 binding motifs, two pleckstrin homology (PH) domains, and a leucine zipper (Flynn et al, 1993). Additionally, AFAP-110 contains a carboxy terminal, actin-binding domain (Qian et al, 2000). These data indicate that AFAP-110 could function by serving to link Src, as well as a variety of other signaling proteins, to actin ®laments.…”

Section: Introductionmentioning

confidence: 99%

“…Biochemically, deletion of the leucine zipper motif enabled AFAP-110 Dlzip to exist as a dimer, based upon gel ®ltration analysis (Baisden et al, 2001). In cells, expression of AFAP-110 Dlzip a ected a signi®cant change in cell morphology and actin ®lament integrity, repositioning actin ®laments into rosette-like structures, not unlike those seen in Src-transformed cells, and inducing lamellipodia formation (Qian et al, 1998(Qian et al, , 2000. Thus, AFAP-110 has an intrinsic capability to alter actin ®lament integrity, which may be revealed by cellular signals which direct changes in its conformation.…”

Section: Introductionmentioning

confidence: 99%

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The intrinsic ability of AFAP-110 to alter actin filament integrity is linked with its ability to also activate cellular tyrosine kinases

et al. 2001

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The actin ®lament-associated protein of 110 kDa (AFAP-110) is a Src binding partner that represents a potential modulator of actin ®lament integrity in response to cellular signals. Previous reports have demonstrated that AFAP-110 is capable of directly binding and altering actin ®laments. Deletion of the leucine zipper motif of AFAP-110 (AFAP-110 Dlzip ) has been shown to induce a phenotype which resembles Srctransformed cells, by repositioning actin ®laments into rosettes. This deletion also mimics a conformational change in AFAP-110 that is detected in Src-transformed cells. The results presented here indicate that unlike AFAP-110, AFAP-110 Dlzip is capable of activating cellular tyrosine kinases, including Src family members, and that AFAP-110 Dlzip itself is hyperphosphorylated. The newly tyrosine phosphorylated proteins and activated Src-family members appear to be associated with actinrich lamellipodia. A point mutation that alters the SH3-binding motif of AFAP-110 Dlzip prevents it from activating tyrosine kinases and altering actin ®lament integrity. In addition, a deletion within a pleckstrin homology (PH) domain of AFAP-110 Dlzip will also revert its e ects upon actin ®laments. Lastly, dominant-positive RhoA V14 will block the ability of AFAP-110 Dlzip from inducing actin ®lament rosettes, but does not inhibit Src activation. Thus, conformational changes in AFAP-110 enable it to activate cellular kinases in a mechanism requiring SH3 and/or PH domain interactions. We hypothesize that cellular signals which alter AFAP-110 conformation, enable it to activate cellular kinases such as cSrc, which then direct changes in actin ®lament integrity in a Rho-dependent fashion. Oncogene (2001) 20, 6607 ± 6616.

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AFAP1L1, a novel associating partner with vinculin, modulates cellular morphology and motility, and promotes the progression of colorectal cancers

et al. 2014

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We have previously identified actin filament-associated protein 1-like 1 (AFAP1L1) as a metastasis-predicting marker for spindle cell sarcomas by gene expression profiling, and demonstrated that AFAP1L1 is involved in the cell invasion process by in vitro analyses. However, its precise molecular function has not been fully elucidated, and it remains unknown whether AFAP1L1 could be a prognostic marker and/or therapeutic target of other malignancies. In this study, we found a marked elevation of AFAP1L1 gene expression in colorectal cancer (CRC) tissues as compared to the adjacent normal mucosa. Multivariate analysis revealed that AFAP1L1 was an independent and significant factor for the recurrence of rectal cancers. Moreover, the addition of the AFAP1L1 expression level to the lymph node metastasis status provided more predictive information regarding postoperative recurrence in rectal cancers. AFAP1L1-transduced CRC cells exhibited a rounded shape, increased cell motility on planar substrates, and resistance to anoikis in vitro. AFAP1L1 localized to the ringed structure of the invadopodia, together with vinculin, and AFAP1L1 was identified as a novel associating partner of vinculin by immunoprecipitation assay. AFAP1L1-transduced cells showed accelerated tumor growth in vivo, presumably reflecting the anoikis resistance of these AFAP1L1-expressing cells. Furthermore, the local administration of a siRNA against AFAP1L1 significantly suppressed the in vivo tumor growth of xenografts, suggesting that AFAP1L1 might be a candidate therapeutic target for CRCs. These results suggest that AFAP1L1 plays a role in the progression of CRCs by modulating cell shape and motility and by inhibiting anoikis, presumably through interactions with vinculin-including protein complexes.

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The Carboxy Terminus of AFAP-110 Modulates Direct Interactions with Actin Filaments and Regulates Its Ability to Alter Actin Filament Integrity and Induce Lamellipodia Formation

Cited by 47 publications

References 33 publications

Conversion of Mechanical Force into Biochemical Signaling

Conversion of Mechanical Force into Biochemical Signaling

The intrinsic ability of AFAP-110 to alter actin filament integrity is linked with its ability to also activate cellular tyrosine kinases

AFAP1L1, a novel associating partner with vinculin, modulates cellular morphology and motility, and promotes the progression of colorectal cancers

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