Src-mediated phosphorylation of mammalian Abp1 (DBNL) regulates podosome rosette formation in transformed fibroblasts

Boateng, Lindsy R.; Cortesio, Christa L.; Huttenlocher, Anna

doi:10.1242/jcs.116038

Cited by 9 publications

(12 citation statements)

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“…Thus, phosphorylation at Thr526 appears to provide a means of regulating proteolysis mediated by the nearby PEST sequences. Since PEST sequences are a conserved feature of Abp1p homologs in fungal and mammalian species and phosphorylation within the PRR is seen in mouse (Larbolette et al 1999;Han et al 2003;Zanivan et al 2008;Larive et al 2009;Boateng et al 2012) and human Abp1 (Olsen et al 2006;Molina et al 2007), we propose that this regulation of Abp1p stability may be a conserved feature of this family. It is possible that the putative intramolecular interaction between the SH3 domain and the PRR, which is supported by our NMR data, is affected by phosphorylation at Thr526 since this site lies between the SH3 domain and the PRR ( Figure 5B).…”

Section: Discussionmentioning

confidence: 89%

“…Since mAbp1 is phosphorylated (Larbolette et al 1999;Han et al 2003;Larive et al 2009;Boateng et al 2012) and several phosphorylated peptides from yeast Abp1p have been identified in large-scale studies (Ficarro et al 2002;Ubersax et al 2003;Holt et al 2009), we sought to further investigate the possible phosphorylation of Abp1p. Interestingly, Abp1p migrates as a double-protein band on SDS-PAGE gels (Drubin et al 1988).…”

Section: Yeast Abp1p Is Phosphorylated Within the Prrmentioning

confidence: 99%

“…A sequence logo, which quantifies conservation at each position in the alignment, is also shown. et al 1999; Han et al 2003;Larive et al 2009;Boateng et al 2012) and phosphorylated peptides from Abp1p have been identified in several global studies of yeast protein phosphorylation (Ficarro et al 2002;Ubersax et al 2003;Chi et al 2007;Holt et al 2009), a role for phosphorylation in the function of Abp1p might be expected.…”

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confidence: 97%

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The Importance of Conserved Features of Yeast Actin-Binding Protein 1 (Abp1p): The Conditional Nature of Essentiality

Garcı́a¹,

Stollar

Davidson

2012

Genetics

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Saccharomyces cerevisiae Actin-Binding Protein 1 (Abp1p) is a member of the Abp1 family of proteins, which are in diverse organisms including fungi, nematodes, flies, and mammals. All proteins in this family possess an N-terminal Actin Depolymerizing Factor Homology (ADF-H) domain, a central Proline-Rich Region (PRR), and a C-terminal SH3 domain. In this study, we employed sequence analysis to identify additional conserved features of the family, including sequences rich in proline, glutamic acid, serine, and threonine amino acids (PEST), which are found in all family members examined, and two motifs, Conserved Fungal Motifs 1 and 2 (CFM1 and CFM2), that are conserved in fungi. We also discovered that, similar to its mammalian homologs, Abp1p is phosphorylated in its PRR. This phosphorylation is mediated by the Cdc28p and Pho85p kinases, and it protects Abp1p from proteolysis mediated by the conserved PEST sequences. We provide evidence for an intramolecular interaction between the PRR region and SH3 domain that may be affected by phosphorylation. Although deletion of CFM1 alone caused no detectable phenotype in any genetic backgrounds or conditions tested, deletion of this motif resulted in a significant reduction of growth when it was combined with a deletion of the ADF-H domain. Importantly, this result demonstrates that deletion of highly conserved domains on its own may produce no phenotype unless the domains are assayed in conjunction with deletions of other functionally important elements within the same protein. Detection of this type of intragenic synthetic lethality provides an important approach for understanding the function of individual protein domains or motifs.

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Section: Discussionmentioning

confidence: 89%

Section: Yeast Abp1p Is Phosphorylated Within the Prrmentioning

confidence: 99%

mentioning

confidence: 97%

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The Importance of Conserved Features of Yeast Actin-Binding Protein 1 (Abp1p): The Conditional Nature of Essentiality

Garcı́a¹,

Stollar

Davidson

2012

Genetics

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“…Cancer Cells-We previously reported that mAbp1 impairs the migration of Src-transformed NIH 3T3 cells (8). To investigate whether mAbp1 also negatively regulates invasion of breast cancer cells, we depleted mAbp1 in MTLn3 and MDA-MB-231 breast cancer cell lines using retroviral shRNA (Fig.…”

Section: Mabp1 Inhibits Invasion Of Breastmentioning

confidence: 99%

“…Both mAbp1 and cortactin have identified roles during endocytosis and vesicle trafficking (3)(4)(5), and both proteins localize to lamellipodia, podosomes, and dorsal ruffles (4,6,7). However, in contrast to cortactin, mAbp1 impairs podosome formation and invasive migration of Src-transformed fibroblasts (8).…”

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confidence: 99%

Mammalian Actin-binding Protein-1/Hip-55 Interacts with FHL2 and Negatively Regulates Cell Invasion

Boateng

Bennin

Oliveira

et al. 2016

Journal of Biological Chemistry

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Mammalian actin-binding protein-1 (mAbp1) is an adaptor protein that binds actin and modulates scission during endocytosis. Recent studies suggest that mAbp1 impairs cell invasion; however, the mechanism for the inhibitory effects of mAbp1 remain unclear. We performed a yeast two-hybrid screen and identified the adaptor protein, FHL2, as a novel binding partner that interacts with the N-terminal actin depolymerizing factor homology domain (ADFH) domain of mAbp1. Here we report that depletion of mAbp1 or ectopic expression of the ADFH domain of mAbp1 increased Rho GTPase signaling and breast cancer cell invasion. Moreover, cell invasion induced by the ADFH domain of mAbp1 required the expression of FHL2. Taken together, our findings show that mAbp1 and FHL2 are novel binding partners that differentially regulate Rho GTPase signaling and MTLn3 breast cancer cell invasion.Invasive migration of cancer cells is dependent on a dynamic actin cytoskeleton (1). Key regulators of the actin cytoskeleton include actin-binding proteins like cortactin, which have been implicated in invadopodia and podosome formation and invasive cell migration. Mammalian actin-binding protein-1 (mAbp1, 2 Hip-55, DBNL) has significant structural homology to cortactin and binds actin with an N-terminal ADFH domain (2). Both mAbp1 and cortactin have identified roles during endocytosis and vesicle trafficking (3-5), and both proteins localize to lamellipodia, podosomes, and dorsal ruffles (4, 6, 7). However, in contrast to cortactin, mAbp1 impairs podosome formation and invasive migration of Src-transformed fibroblasts (8).To determine how mAbp1 regulates cell invasion, we performed a yeast two-hybrid screen and identified the fourand-a-half LIM domain protein 2 (FHL2) as a novel mAbp1-binding partner that does not interact with cortactin. FHL2 (also known as DRAL or SLIM3) is a LIM domain protein consisting of four-and-a-half LIM domains. Each LIM domain contains two zinc finger loops that mediate proteinprotein interactions. FHL2 interacts with Ͼ50 proteins (9) including integrins and focal adhesion kinase (FAK) (10, 11), actin (12), and the transcription factors estrogen receptor (ER), activator protein-1 (AP-1), and ␤-catenin (13-18). Interestingly, FHL2 is expressed at low levels in many tissues like breast, placenta, uterus, and the lungs (17, 19) and is up-regulated in cancer. For example, FHL2 expression is increased in many cancers including breast (20, 21), ovarian (22), prostate (23), lung (24), colon (25), brain (26), and skin cancers (27). In primary breast tumors, patients with high levels of FHL2 have a poorer survival rate (21), suggesting that FHL2 may be involved in cancer progression. Furthermore, overexpression of FHL2 in MDA-MB-231 human breast cancer cells enhances colony formation in soft agar, whereas knockdown reduces the ability to form colonies (20).Here we report a novel interaction between the N-terminal ADFH domain of mAbp1 and FHL2 that modulates cell invasion. In the yeast, but not the mammalian form, the N terminu...

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Src‐dependent Tks5 phosphorylation regulates invadopodia‐associated invasion in prostate cancer cells

et al. 2013

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BACKGROUND The Src tyrosine kinase substrate and adaptor protein Tks5 had previously been implicated in the invasive phenotype of normal and transformed cell types via regulation of cytoskeletal structures called podosomes/invadopodia. The role of Src-Tks5 signaling in invasive prostate cancer, however, had not been previously evaluated. METHODS We measured the relative expression of Tks5 in normal (n = 20) and cancerous (n = 184, from 92 patients) prostate tissue specimens by immunohistochemistry using a commercially available tumor microarray. We also manipulated the expression and activity of wild-type and mutant Src and Tks5 constructs in the LNCaP and PC-3 prostate cancer cell lines in order to ascertain the role of Src-Tks5 signaling in invadopodia development, matrix-remodeling activity, motility, and invasion. RESULTS Our studies demonstrated that Src was activated and Tks5 upregulated in high Gleason score prostate tumor specimens and in invasive prostate cancer cell lines. Remarkably, overexpression of Tks5 in LNCaP cells was sufficient to induce invadopodia formation and associated matrix degradation. This Tks5-dependent increase in invasive behavior further depended on Src tyrosine kinase activity and the phosphorylation of Tks5 at tyrosine residues 557 and 619. In PC-3 cells we demonstrated that Tks5 phosphorylation at these sites was necessary and sufficient for invadopodia-associated matrix degradation and invasion. CONCLUSIONS Our results suggest a general role for Src-Tks5 signaling in prostate tumor progression and the utility of Tks5 as a marker protein for the staging of this disease.

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Src-mediated phosphorylation of mammalian Abp1 (DBNL) regulates podosome rosette formation in transformed fibroblasts

Cited by 9 publications

References 0 publications

The Importance of Conserved Features of Yeast Actin-Binding Protein 1 (Abp1p): The Conditional Nature of Essentiality

The Importance of Conserved Features of Yeast Actin-Binding Protein 1 (Abp1p): The Conditional Nature of Essentiality

Mammalian Actin-binding Protein-1/Hip-55 Interacts with FHL2 and Negatively Regulates Cell Invasion

Src‐dependent Tks5 phosphorylation regulates invadopodia‐associated invasion in prostate cancer cells

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