Induction of Apoptosis after Expression of PYK2, a Tyrosine Kinase Structurally Related to Focal Adhesion Kinase

Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell–ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase β (CAKβ) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFα and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH₂-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.     

Characterization of mouse integrin alpha3 subunit gene.

Integrin alpha3beta1 (VLA-3) is an adhesion receptor for extracellular matrix proteins including various isoforms of laminin. We have isolated mouse genomic clones encoding the integrin alpha3 subunit and deduced the exon/intron organization. The mouse integrin alpha3 subunit gene is encoded by 26 exons spanning 40 kb. The exon/intron structure of the integrin alpha3 subunit gene resembles that of the integrin alpha6 subunit gene, but differs somewhat from those of other members of the integrin family. We have demonstrated that the cytoplasmic domain splicing variants of the alpha3 subunits (alpha3A and alpha3B) are generated by alternative exon usage. We also cloned the 5'-flanking region and performed a preliminary analysis of its promoter activity in various tumor cell lines with different degrees of integrin alpha3 expression. Following transfection, activity in the luciferase assay was found to be roughly correlated with the expression level of integrin alpha3 as measured by flow cytometry. Furthermore, the luciferase assay was performed with normal and SV-40- or polyoma virus-transformed fibroblasts. In mouse, human, and hamster fibroblasts, higher levels of luciferase expression were observed in transformed cells than in normal cells. This result is consistent with our previous finding that integrin alpha3 expression at both the protein and mRNA levels is enhanced upon oncogenic transformation of fibroblasts by tumor viruses.     

Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation

Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.     

Diverse effects of RacV12 on cell transformation by Raf: partial inhibition of morphological transformation versus deregulation of cell cycle control

Activated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation. This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins. NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1-oestrogen receptor fusion protein (c-Raf-1-BxB-ER, N-BxB-ER cells) undergo morphological transformation upon stimulation of the Raf kinase. We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf. Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells. Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho. With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac. Another feature of cell transformation is the deregulation of cell cycle control. NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ER protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase. Our results show that in N-BxB-ER-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21(Cip1). These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control.     

Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover

Cell-to-extracellular matrix adhesion is regulated by a multitude of pathways initiated distally to the core cell–matrix adhesion machinery, such as via growth factor signaling. In contrast to these extrinsically sourced pathways, we now identify a regulatory pathway that is intrinsic to the core adhesion machinery, providing an internal regulatory feedback loop to fine tune adhesion levels. This autoinhibitory negative feedback loop is initiated by cell adhesion to vitronectin, leading to PAK4 activation, which in turn limits total cell–vitronectin adhesion strength. Specifically, we show that PAK4 is activated by cell attachment to vitronectin as mediated by PAK4 binding partner integrin αvβ5, and that active PAK4 induces accelerated integrin αvβ5 turnover within adhesion complexes. Accelerated integrin turnover is associated with additional PAK4-mediated effects, including inhibited integrin αvβ5 clustering, reduced integrin to F-actin connectivity and perturbed adhesion complex maturation. These specific outcomes are ultimately associated with reduced cell adhesion strength and increased cell motility. We thus demonstrate a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion.     

Cell-matrix entanglement and mechanical anchorage of fibroblasts in three-dimensional collagen matrices

Fibroblast-3D collagen matrix culture provides a physiologically relevant model to study cell–matrix interactions. In tissues, fibroblasts are phagocytic cells, and in culture, they have been shown to ingest both fibronectin and collagen-coated latex particles. Compared with cells on collagen-coated coverslips, phagocytosis of fibronectin-coated beads by fibroblasts in collagen matrices was found to be reduced. This decrease could not be explained by integrin reorganization, tight binding of fibronectin beads to the collagen matrix, or differences in overall bead binding to the cells. Rather, entanglement of cellular dendritic extensions with collagen fibrils seemed to interfere with the ability of the extensions to interact with the beads. Moreover, once these extensions became entangled in the matrix, cells developed an integrin-independent component of adhesion. We suggest that cell–matrix entanglement represents a novel mechanism of cell anchorage that uniquely depends on the three-dimensional character of the matrix.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

Amygdalin Influences Bladder Cancer Cell Adhesion and Invasion In Vitro

The cyanogenic diglucoside amygdalin, derived from Rosaceae kernels, is employed by many patients as an alternative anti-cancer treatment. However, whether amygdalin indeed acts as an anti-tumor agent is not clear. Metastasis blocking properties of amygdalin on bladder cancer cell lines was, therefore, investigated. Amygdalin (10 mg/ml) was applied to UMUC-3, TCCSUP or RT112 bladder cancer cells for 24 h or for 2 weeks. Tumor cell adhesion to vascular endothelium or to immobilized collagen as well as tumor cell migration was examined. Effects of drug treatment on integrin α and β subtypes, on integrin-linked kinase (ILK) and total and activated focal adhesion kinase (FAK) were also determined. Integrin knock-down was carried out to evaluate integrin influence on migration and adhesion. A 24 h or 2 week amygdalin application distinctly reduced tumor cell adhesion and migration of UMUC-3 and RT112 cells. TCCSUP adhesion was also reduced, but migration was elevated under amygdalin. Integrin subtype expression was significantly and specifically altered by amygdalin depending on the cell line. ILK was moderately, and activated FAK strongly, lost in all tumor cell lines in the presence of amygdalin. Knock down of β1 integrin caused a significant decrease in both adhesion and migration of UMUC-3 cells, but a significant increase in TCCSUP adhesion. Knock down of β4 integrin caused a significant decrease in migration of RT112 cells. Since the different actions of amygdalin on the different cell lines was mirrored by β1 or β4 knock down, it is postulated that amygdalin influences adhesion and migratory properties of bladder cancer cells by modulating β1 or β4 integrin expression. The amygdalin induced increase in TCCSUP migratory behavior indicates that any anti-tumor benefits from amygdalin (seen with the other two cell lines) may depend upon the cancer cell type.     

Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg²⁺ or Mn²⁺ but not Ca²⁺. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ~110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.     

Myofibroblasts protect myoblasts from intrinsic apoptosis associated with differentiation via β1 integrin‐PI3K/Akt pathway

Skeletal myoblasts withdrawing from cell cycle is a prerequisite for myodifferentiation, while upon proliferation/differentiation transformation, a large portion of myoblasts will undergo apoptosis. Skeletal fibroblasts, residing in muscle tissue both during and post myogenesis, have been proofed to play pivotal roles in muscle development, while their effect on myoblast apoptosis being coincident with differentiation has not been reported. Using a membrane insert co‐culture system, we studied it and found that the mitochondrial pathway played a crucial role in myoblast apoptosis during differentiation, and fibroblasts promoted not only cell cycle withdrawal but also myoblast survival in a paracrine fashion, which was coupled with upregulations of β1 integrin, phosphorylated Akt and anti‐apoptotic protein Bcl2. To determine the effect of β1 integrin in the process, we transfected myoblasts with siRNA specific for β1 integrin before co‐culture and found that β1 integrin knockdown abolished anti‐apoptotic ability of myoblasts and inhibited Akt activation and Bcl2 expression. Blockage of PI3K/Akt pathway with wortmannin also seriously impaired the protective effect of fibroblasts on myoblasts and fibroblast‐induced Bcl2 expression. The data demonstrated that fibroblasts protected myoblasts from intrinsic apoptosis associated with differentiation, and β1 integrin‐PI3K/Akt pathway activation was required for the process.     

Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.     

An Elmo–Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion

Cell–cell contact formation is a dynamic process requiring the coordination of cadherin-based cell–cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell–cell adhesion identified an Elmo–Dock complex. This was unexpected as Elmo–Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell–cell contacts in Madin–Darby canine kidney cells. At cell–cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell–cell adhesion. Upon completion of cell–cell adhesion, Elmo2 and Dock1 no longer localize to cell–cell contacts and are not required subsequently for the maintenance of cell–cell adhesion. These studies show that Elmo–Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell–cell adhesion.     

DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals

Death-associated protein kinase (DAP-kinase) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptosis systems. However, its apoptosis-promoting mechanism is poorly understood. Here, we demonstrate that DAP-kinase suppresses integrin-mediated cell adhesion and signal transduction, whereas dominant-negative interference of this kinase promotes adhesion. This effect of DAP-kinase is neither a consequence of apoptosis nor a result of decreased expression of integrins. Rather, DAP-kinase downregulates integrin activity through an inside-out mechanism. We present evidence indicating that this adhesion-inhibitory effect accounts for a major mechanism of the apoptosis induced by DAP-kinase. First, in growth-arrested fibroblasts, DAP-kinase triggers apoptosis in cells plated on fibronectin, but does not affect the death of cells on poly-l-lysine. Second, in epithelial cells, DAP-kinase induces apoptosis in the anoikis-sensitive MCF10A cells, but not in the anoikis-resistant BT474 cells. Most importantly, the apoptosis-promoting effect of DAP-kinase is completely abolished by enforced activation of integrin-mediated signaling pathways from either integrin itself or its downstream effector, FAK. Finally, we show that integrin or FAK activation blocks the ability of DAP-kinase to upregulate p53. Our results indicate that DAP-kinase exerts apoptotic effects by suppressing integrin functions and integrin-mediated survival signals, thereby activating a p53-dependent apoptotic pathway.     

β3 integrin–mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner

Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin–dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2–induced Smad signaling through a Cdc42–Src–FAK–ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine.     

Fibronectin protects prostate cancer cells from tumor necrosis factor-alpha-induced apoptosis via the AKT/survivin pathway

Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84--> Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via beta1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-alpha-induced apoptosis. Evidence is provided that expression of beta1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-alpha-induced apoptosis. In contrast, the beta1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-alpha in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis.     

Gab1 is required for cell cycle transition, cell proliferation, and transformation induced by an oncogenic met receptor

We have shown previously that either Grb2- or Shc-mediated signaling from the oncogenic Met receptor Tpr-Met is sufficient to trigger cell cycle progression in Xenopus oocytes. However, direct binding of these adaptors to Tpr-Met is dispensable, implying that another Met binding partner mediates these responses. In this study, we show that overexpression of Grb2-associated binder 1 (Gab1) promotes cell cycle progression when Tpr-Met is expressed at suboptimal levels. This response requires that Gab1 possess an intact Met-binding motif, the pleckstrin homology domain, and the binding sites for phosphatidylinositol 3-kinase and tyrosine phosphatase SHP-2, but not the Grb2 and CrkII/phospholipase Cγ binding sites. Importantly, we establish that Gab1-mediated signals are critical for cell cycle transition promoted by the oncogenic Met and fibroblast growth factor receptors, but not by progesterone, the natural inducer of cell cycle transition in Xenopus oocytes. Moreover, Gab1 is essential for Tpr-Met–mediated morphological transformation and proliferation of fibroblasts. This study provides the first evidence that Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation. This study identifies Gab1 and its associated signaling partners as potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor.     

The Arf tumor suppressor protein inhibits Miz1 to suppress cell adhesion and induce apoptosis

Oncogenic stress induces expression of the alternate reading frame (Arf) tumor suppressor protein. Arf then stabilizes p53, which leads to cell cycle arrest or apoptosis. The mechanisms that distinguish both outcomes are incompletely understood. In this study, we show that Arf interacts with the Myc-associated zinc finger protein Miz1. Binding of Arf disrupts the interaction of Miz1 with its coactivator, nucleophosmin, induces the sumoylation of Miz1, and facilitates the assembly of a heterochromatic complex that contains Myc and trimethylated H3K9 in addition to Miz1. Arf-dependent assembly of this complex leads to the repression of multiple genes involved in cell adhesion and signal transduction and induces apoptosis. Our data point to a tumor-suppressive pathway that weakens cell–cell and cell–matrix interactions in response to expression of Arf and that may thereby facilitate the elimination of cells harboring an oncogenic mutation.     

Site-specific Phosphorylation of Kindlin-3 Protein Regulates Its Capacity to Control Cellular Responses Mediated by Integrin αIIbβ3

The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins to their cytoplasmic tails. Kindlins are integrin cytoplasmic tail binding partners and are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin α_IIbβ₃, to investigate whether phosphorylation of kindlin-3 regulates integrin activation. When HEL cells were stimulated with thrombopoietin or phorbol 12-myristate 13-acetate (PMA), α_IIbβ₃ became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of β₃ integrin and kindlin-3 in focal adhesions, and enhanced β₃ integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr⁴⁸² or Ser⁴⁸⁴ was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476–485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin β₃ activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs.