Phosphatidylinositol Phosphate 5-Kinase Iγi2 in Association with Src Controls Anchorage-independent Growth of Tumor Cells*

Background: PIPKIγ isoforms play roles in cell migration, polarization, and membrane trafficking and are overexpressed in triple-negative breast cancers, indicating protumorigenic functions. Results: PIPKIγi2 associates with the C terminus of Src, and this interaction interdependently controls their functioning. Conclusion: PIPKIγi2 and Src synergistically control anchorage-independent tumor cell growth. Significance: This study shows unexpected mechanisms for a phosphatidylinositol 4,5-biphosphate-generating enzyme that synergizes with the proto-oncogene Src to regulate oncogenic signaling. A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.

The ability of tumor cells to defy anoikis, cell death caused by lack of cell-matrix interaction, and grow in an anchorage-independent manner determines their capacity to survive in the vasculature and lymphatic circulation during tumor metastasis (1,2). In adherent cells, focal adhesions are the contact points of cells to their underlying substratum and also serve as signaling hubs. Anchorage dependence of normal cells stems from the fact that they derive a large part of their proliferative and survival signals from their substratum via focal adhesions (3). Contradicting this, many focal adhesion proteins, including FAK, integrin-linked kinase, paxillin, Src, talin, and pCAS130, are actively involved in oncogenic processes that allow tumor cells to survive/grow in an anchorage-independent manner and promote tumorigenesis (4 -7). However, the precise mechanism for how tumor cells assemble the signaling molecules at the plasma membrane following the disruption of cell-matrix interaction, thus bestowing anchorage independence for survival and growth, remains poorly understood (8 -11).
Src, non-receptor tyrosine kinase and proto-oncogene, regulates the PIPKI␥i2 interaction with talin (28). Src activation is a hallmark of many cancers, and several mechanisms are implicated in its activation of tumorigenic processes (29,30). The plasma membrane recruitment and activation of Src is primarily mediated by myristolylation of glycine residue in its N terminus (31), although highly conserved basic residues in its N-terminal part also play an important role in Src function and its plasma membrane recruitment via electrostatic interaction with anionic lipid molecules (32). Here, we show that PIPKI␥i2 and Src, both focal adhesion molecules, form a signaling complex following the disruption of cell-matrix interaction and support the anchorage-independence of tumor cells.
DNA Constructs, Mutagenesis, and siRNA-PIPKI␥ isoforms or PIPKI␥i2 mutants were subcloned into MluI and SalI sites in frame with HA tag in the N terminus of PWPT vector (Addgene) as described previously (22). Full-length chicken Src or Src mutants used in the study were cloned into BamHI and SalI sites of PWPT vector. All the mutations used in the study were created using a QuikChange site-directed mutagenesis kits (Stratagene) followed by DNA sequencing to confirm the integrity of the DNA sequence. For cloning the C terminus of Src, oligonucleotides used for annealing were: TCGAGGAGG-ACTACTTCACGTCCACCGAGCCCCAGTACCAGCCCG-GGGAGAACCTCTAGG (sense) and TCGACCTAGAGGTT-CTCCCCGGGCTGGTACTGGGGCTCGGTGGACGTG-AAGTAGTCCTCC (antisense). After annealing of these oligonucleotides, 5Ј-prime and 3Ј-prime ends harbor XhoI and SalI sites, respectively, for cloning into pEGFP-C3 vector (Clontech) in frame with GFP in the N terminus. This construct was further subcloned into PWPT-GFP vector for retroviral infection.
Cell Culture-MDA-MB-231, NIH3T3, HEK293, and HEK293FT cells were cultured in DMEM containing 10% FBS. T47D and HCC1954 cells were cultured in RMPI 1640 containing 10% FBS. SUM1315 cells were culture in Ham's F12 supplemented with 5% FCS. Suspended cells in the study refer to the cells resuspended in medium containing 0.1% BSA and 1.0% FBS after trypsinization/detachment and incubated at 37°C in the incubator for 2-3 h except for time course study. For overnight culture in suspension condition, cells suspended in the medium were seeded into the culture plate coated with 0.3% agar to avoid cell attachment. For stimulation of cells in adherent condition, cells were serum-starved overnight before stimulating the cells with 10% FBS or EGF (50 g/ml) for the indicated time periods. For the stimulation of cells in suspension condition, cells were resuspended as described above and incubated for 2-3 h before stimulating with FBS (10% FBS) or EGF (50 g/ml) or extracellular matrix protein (combination of fibronectin/ collagen type I, 25 g/ml each) for the indicated time periods.
Transfection or Lentiviral Infection-For siRNA-mediated knockdown of genes, Lipofectamine RNAiMAX (Invitrogen) was used following the protocol provided by manufacturer, and cells were used 48 -72 h post-transfection. For transient transfection into HEK293 cells, Lipofectamine 2000 (Invitrogen) was used. Cells were harvested 24 hours post-transfection. For the expression or co-expression of genes into MDA-MB-231 or NIH3T3 or MCF-7 cells, the lentiviral system was used as described previously (22). Cells were harvested 24 -48 h post-infection (70 -80% infection efficiency were achieved for the experiments).
Immunoprecipitation and Immunoblotting-Cells were lysed using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 10 mM NaF, 5 mM Na 3 VO 4 , and protease inhibitors). Clear supernatants were incubated with indicated antibodies for 3-4 h to overnight at 4°C followed by isolation of immune complexes using protein G-Sepharose 4B beads (Amersham Biosciences). The beads were washed three times with lysis buffer before eluting the immune complexes with 2ϫ sample buffer and then subjected to immunoblotting using indicated antibodies.
GST Pulldown Assay-Different regions of chicken Src were PCR-amplified and cloned into pGEX-6P-1 (Novagen). Proteins were expressed into BL21 and purified using glutathione-Sepharose 4B beads (Amersham Biosciences). For in vitro binding study, purified GST fusion proteins immobilized on the Sepharose beads were incubated with His-tagged PIPKI␥i2 purified from bacteria or with cell lysates prepared from HEK293 cells transfected with HA-PIPKI␥i2 at 4°C for 1 h followed by elution of bound proteins with 2ϫ sample buffer for immunoblotting.
Cell Proliferation and Anchorage-independent Growth-For cell proliferation assay, MDA-MB-231 cells were seeded into 12-well culture plate (1,000 cells/well) in DMEM containing 10% FBS. Cells were manually counted every second day for up to 8 days.
For anchorage-independent growth, cells were suspended in medium containing 0.3% agar and seeded into 24-well culture plates. To avoid cell attachment, culture plates were precoated with 0.5% agar before cell seeding. Cultures were fed with fresh medium in every 3-5 days and cultured for 10 -28 days depending upon the cells type used. Similarly, cell numbers used for seeding were also adjusted depending upon the efficiency of cells to form colonies. In some cases, Src inhibitor (PP1, 0.5 M) was added into the medium. Colonies developed were fixed with 3.7% paraformaldehyde and stained with 0.1% crystal violet to facilitate the visualization and counting.
Immunofluorescence Microscopy (IF)-For examining the colocalization of PIPKI␥i2 and Src at focal adhesions, cells were seeded into collagen type I-or fibronectin-coated coverslip and incubated for 30 min before fixing the cells with 3.7% paraformaldehyde. Cells were permeabilized with 0.1% Triton X-100 before blocking with 3% BSA in PBS. The same procedures were used for IF study of the colonies developed in the soft agar. Cells were incubated with primary antibody overnight at 4°C followed by incubation with Alexa 555-and/or Alexa 488-conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. Slides were mounted using Vectashield and visualized with a Nikon TE2000-U microscope using 63ϫ objective lenses. The images were acquired using MetaMorph and processed using adobe Photoshop.
For examining the phosphatidylinositol 4,5-biphosphate distribution in the PIPKI␥ or PIPKI␥i2 knockdown cells, MDA-MB-231 cells were transfected with siRNA as described above. After 24 -36 h, cells were retransfected with plasmids for the expression of GFP-PLC␦-PH or GFP-PLC␦-PH mutant. Cells were processed for IF study following the overnight culture.
Statistical Analysis-The data are presented as means Ϯ S.D. from at least three-independent experiments. Unpaired t test was conducted to determine the p value, and the statistical significance between two groups (p value equal to or less than 0.05 were considered significant).

RESULTS
PIPKI␥/PIPKI␥i2 Regulate the Anchorage-independent Growth of Tumor Cells-PIPKI␥i2 is a phosphatidylinositol 4,5-biphosphate-generating enzyme targeted to cell-matrix interaction sites via an interaction with talin (33,35). Src phosphorylation of tyrosine residues at the C terminus of PIPKI␥i2 (red Tyr residues in Fig. 1A) regulates its interaction with talin (28). Increased expression of PIPKI␥ in breast cancer tissues inversely correlates with patient survival, indicating its potential role in tumor progression (25). To define an oncogenic role for PIPKI␥, pan-PIPKI␥ or PIPKI␥i2 was knocked down, and the effect on anchorage-independent growth of breast cancer cell lines in soft agar was examined. The knockdown of endogenous PIPKI␥ or PIPKI␥i2 significantly impaired the ability of MDA-MB-231, SUM1315, and T47D cells to grow in an anchorage-independent manner (Fig. 1, B-D). However, cell proliferation was not affected by PIPKI␥i2 knockdown but was affected by pan-PIPKI␥ knockdown in MDA-MB-231 cells in two-dimensional culture (Fig. 1E). Consistently, the loss of PIPKI␥i2 was not sufficient to affect the localization of GFP-PLC␦-PH, a biosensor of phosphatidylinositol 4,5-biphosphate, whereas pan-PIPKI␥ knockdown resulted in impaired localization of GFP-PLC␦-PH in the plasma membrane (Fig. 1F). These results indicate the collective function of different PIPKI␥ variants in phosphatidylinositol 4,5-biphosphate synthesis in the plasma membrane. Furthermore, the knockdown of endogenous PIPKI␥i2 in MDA-MB-231 cells expressing siRNA-resistant PIPKI␥i2 did not affect the anchorageindependent growth (Fig. 1G). Further, the kinase-dead mutant of PIPKI␥i2 poorly rescued anchorage-independent growth, signifying the importance of kinase activity. The expression level of ectopically expressed siRNA-resistant PIPKI␥i2 was severalfold higher than that of endogenous PIPKI␥i2 and resulted in induced anchorage-independent growth. To investigate the role of each PIPKI␥ splice variant, PIPKI␥ variants were ectopically expressed into MDA-MB-231 cells, which express a low level of PIPKI␥/PIPKI␥i2 compared with other breast cancer cell lines examined (not shown). As shown in Fig. 2 (A-C), the expression of each variant significantly promoted anchorage-independent growth, although PIPKI␥i2 expression had substantially greater effect, which correlated with its expression level (Fig. 2D). The knockdown of ectopically expressed PIPKI␥i2 completely abrogated the growth promoted by PIPKI␥i2 overexpression (Fig. 2E). Moreover, PIPKI␥i1 or PIPKI␥i2 expression into MDA-MB-231 cells did not show an obvious effect on cell proliferation in two-dimensional culture (Fig. 2F), indicating specificity for anchorage independent growth regulation. PIPKI␥i2 overexpression also promoted anchorage-independent growth of MCF-7 cells (Fig. 2G).
PIPKI␥i2 Regulates Src Activation Downstream of Growth Factor Receptors and Integrins-To delineate the signaling molecules involved in PIPKI␥ regulation of anchorage-indepen-dent growth, we examined the impact of PIPKI␥ or PIPKI␥i2 knockdown on Src, a key signaling molecule with roles in cell survival, oncogenic, and/or anchorage-independent growth (4, 6, 29) that phosphorylates PIPKI␥i2 (28). Suspension culture of different tumor cells displayed significantly impaired Src activation (tyrosine phosphorylated Src in its activation site) upon PIPKI␥ or PIPKI␥i2 knockdown (Fig. 3, A and B, and data not shown). Corroborating this, the overexpression of PIPKI␥i2 increased the activation level of Src, without a noticeable change in FAK activation (assessed by autophosphorylation of FAK at Tyr 397 ) (Fig. 3C). The knockdown of ectopically expressed PIPKI␥i2 abrogated activation level of Src (Fig. 3D), and this coincided with significantly reduced anchorage-independent growth (Fig. 2E). However, the impact of PIPKI␥/ PIPKI␥i2 knockdown or overexpression on Src activation was less obvious in the adherent condition (not shown).
Src Is Required for PIPKI␥i2-induced Anchorage-independent Growth and Vice Versa-In support of the above results, the knockdown of endogenous Src or the use of a Src inhibitor blocked the PIPKI␥i2-induced anchorage-independent growth (Fig. 4, A-D), indicating the role for Src in PIPKI␥i2-regulation of oncogenic growth. These results are consistent with a key role for Src in mediating cell survival and growth in both cellular and in vivo systems (6,36).
After demonstrating the Src function in PIPKI␥i2-induced anchorage-independent growth, we inquired whether PIPKI␥/ PIPKI␥i2 is required for Src function. A feature of Src-transformed cells is a disorganized cytoskeleton with multiple cell protrusions (37). Consistently, the knockdown of PIPKI␥i2, but not PIPKI␣, abrogated the disorganized actin cytoskeleton phenotype induced by Src expression in MDA-MB-231 cells (Fig. 4E, left panels). Ectopically expressed Src localized to cell protrusions, and this targeting was also significantly reduced by PIPKI␥ or PIPKI␥i2 knockdown (Fig. 4E, right  panels). Similarly, tyrosine phosphorylation of FAK and cortactin, Src substrates, induced by Src expression was reduced upon PIPKI␥i2 knockdown (Fig. 4F). Decreased anchorageindependent growth of Src-expressing cells upon PIPKI␥ or PIPKI␥i2 loss was also accompanied by reduced Src activa- . E, siRNA was used to knock down PIPKI␥i2 or PIPKI␣ from MDA-MB-231 cells transfected with Src. Cells were fixed for IF study to examine the actin cytoskeleton (left panels) and Src localization (right panels). Scale bar, 20 M. F, cortactin and FAK were immunoprecipitated from mock and Src-infected cells after siRNA transfection for PIPKI␥i2 knockdown. Cells were harvested 48 hours post-transfection to immunoprecipitate the endogenous cortactin and FAK followed by immunoblotting using phosphotyrosine antibody. G, siRNA was used for PIPKI␥i2 knockdown in MDA-MB-231 cells infected with lentivirus for Src overexpression. Cells were cultured in soft agar for 2 weeks before counting the colonies. tion (Fig. 4G), indicating that PIPKI␥i2 is required for Src activation and function.
PIPKI␥i2 and Src Synergistically Induce Anchorage-independent Growth-After demonstrating the Src requirement for PIPKI␥i2-induced anchorage-independent growth and vice versa, we examined the ability of PIPKI␥i2 (and other PIPKI␥ variants) to induce oncogenic growth of the nontransformed NIH3T3 or MCF10A cells. Independent expression of PIPKI␥i2 or Src poorly induced the anchorage-independent growth of NIH3T3 cells (Fig. 5A). Strikingly, co-expression of PIPKI␥i2 and Src dramatically increased the anchorage-independent growth. The synergistic effect of PIPKI␥i2 and Src was further demonstrated in MDA-MB-231 cells (Fig. 5B). Among PIPKI␥ variants, PIPKI␥i2 showed the most potent effect, emphasizing the functional specificity of PIPKI␥i2 and Src in anchorageindependent growth regulation (not shown). However, a kinase dead mutant of PIPKI␥i2 poorly induced anchorage-independent growth in synergy with Src, indicating the role of kinase activity of PIPKI␥i2 enzyme (Fig. 5C). Furthermore, an analysis of the expression of PIPKI␥/PIPKI␥i2 demonstrated the link between PIPKI␥/PIPKI␥i2 expression and Src activation in many breast cancer cell lines examined (data not shown). In all tumor cell lines examined, the loss of PIPKI␥/PIPKI␥i2 and/or Src inhibited oncogenic growth on soft agar (Fig. 5, D and E).

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revealed extensive co-localization of endogenous active Src with PIPKI␥i2, predominantly at focal adhesions (Fig. 6A, upper panels). Phosphatidylinositol 4,5-biphosphate-generating enzymes regulate intracellular vesicle trafficking and targeting of signaling molecules to the plasma membrane at sites of adhesion (40,41). PIPKI␥i2 knockdown modestly affected active Src localization at focal adhesions (Fig. 6, A,  lower panels, and B). However, the loss of talin resulted in a more profound defect on Src localization at focal adhesions (Fig. 6, A and B) and is consistent with the role of talin in focal adhesion assembly.
In three-dimensional suspension culture, PIPKI␥i2 knockdown impaired both Src and talin localization at plasma membrane (Fig. 6C). Src extensively co-localized with PIPKI␥i2 at cell-cell contact sites at the plasma membrane (Fig. 6D), whereas other PIPKI␥ variants deficient in focal adhesion targeting (PIPKI␥i1) were poorly localized with Src at plasma membrane. A PIPKI␥i2 mutant deficient in Src phosphorylation and talin binding, thus defective in focal adhesion localization were also poorly co-localized with Src at plasma membrane in suspension culture (not shown). In suspension condition, PIPKI␥i2 forms a stable complex with talin that is promoted by Src expression and PIPKI␥i2 phosphorylation (Fig. 6E). All of these results indicate that PIPKI␥i2 in coordination with talin regulates Src localization at focal adhesions and the plasma membrane in three-dimensional culture. Furthermore, impaired anchorage-independent growth in PIPKI␥i2overexpressing cells after talin knockdown (Fig. 6F) supports the coordinated roles of the focal adhesion molecules, PIPKI␥i2, Src, and talin, in oncogenic signaling.
An Interaction between PIPKI␥i2 and Src Is Required for Anchorage-independent Growth-PIPKI␥i2 is directly phosphorylated by Src (28). PIPKIs often associate with proteins they regulate (12,20,22) as such an interaction between PIPKI␥/ PIPKI␥i2 and Src was explored. The interaction between PIPKI␥ variants and Src was observed in their endogenous levels and after co-expression and co-immunoprecipitation (Fig. 7A).
To define whether PIPKI␥ directly interacts with Src and to determine the Src region required for PIPKI␥i2 interaction, the GST pulldown assays were performed using recombinant GST fusion proteins of Src (kinase domain of Src was not soluble) and His-tagged PIPKI␥i2 or cell lysates prepared from HEK293 cells transfected with HA-PIPKI␥i2 (Fig. 7B). The full-length Src bound with His-tagged PIPKI␥i2 or HA-PIPKI␥i2, indicating that either the kinase domain or the C terminus mediates the Src interaction with PIPKI␥i2. Co-expression and co-immunoprecipitation studies demonstrated that the Src C-termi-nal deletion mutant was defective in PIPKI␥i2 binding (Fig. 7C), indicating that the C terminus is necessary for this interaction. The constitutively active Src (Y527F mutation) showed a modest increase in PIPKI␥i2 interaction (Fig. 7C). Csk kinase that phosphorylates Src in Tyr 527 , promoting an autoinhibitory intramolecular interaction and Src inactivation, binds to C terminus of Src (29). However, PIPKI␥ expression did not affect the Src association with Csk nor Tyr 527 phosphorylation of Src (not shown).
Deletion of the Src C terminus leads to constitutive activation of Src (42). Remarkably, the co-expression of the C-terminal deletion mutant of Src with PIPKI␥i2 failed to induce anchorage-independent growth in synergy with Src (Fig. 7D), indicating the importance of the PIPKI␥i2 interaction with Src. Further, the expression of the GFP fusion protein with C-terminal tail of Src (GFP-C-tail) inhibited the localization of active Src at focal adhesions (not shown) and PIPKI␥i2 interaction with Src (Fig. 7E). It also inhibited the anchorage-independent growth induced by co-expression of PIPKI␥i2 and Src (Fig. 7F). Lower panels, in vitro binding study performed using GST fusion proteins of Src and His-tagged PIPKI␥i2 or cell lysates prepared from HA-PIPKI␥i2 expressing cells. Bound proteins were examined by immunoblotting. C, HEK293 cells were co-transfected with HA-PIPKI␥i2 and Src or its deletion mutants. Co-immunoprecipitation of PIPKI␥i2 with Src and its mutant forms was examined by immunoblotting using anti-HA antibody. N-term., N-terminal; C-term., C-terminal. D, MDA-MB-231 cells expressing either PIPKI␥i2 or Src or mutant Src alone or co-expressing them were culture in soft agar for 10 -12 days before counting the colonies. E, MDA-MB-231 cells co-expressing PIPKI␥i2 and Src were infected with lentivirus for the expression of GFP or GFP-C-tail of Src followed by immunoprecipitation of PIPKI␥i2 to examine the co-immunoprecipitation of Src. F, these cells were cultured in soft agar for 10 -12 days before counting the colonies. All the values are means Ϯ S.D. from three-independent experiments (p values are indicated). NS, nonsignificant p value. NOVEMBER 29, 2013 • VOLUME 288 • NUMBER 48

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The highly conserved basic residues in the N terminus of Src play an important role in Src function and its recruitment to plasma membrane via electrostatic interaction with anionic phospholipids, including phosphatidylinositol 4,5-biphosphate and others (32). However, the precise role of phosphatidylinositol 4,5-biphosphate in Src function is not defined. As shown in Fig. 8A, mutations of all of these basic residues to neutral amino acids impaired Src association with PIPKI␥i2 (Fig. 8B) and the ability of Src to induce oncogenic growth in synergy with PIPKI␥i2 (Fig. 8C). Furthermore, kinase dead PIPKI␥i2 showed impairment in inducing anchorage-independent growth in synergy with Src (Fig. 5C). Taken together, these results indicate a coordinated role of PIPKI␥i2 and Src and phosphatidylinositol 4,5-biphosphate generation in oncogenic signaling and anchorage-independent growth (illustrated in schematic diagram in Fig. 8D).

DISCUSSION
The ability to grow in an anchorage-independent manner is one of the fundamental properties of tumor cells and is a key for metastasis, although the underlying mechanisms are poorly understood. Here, we show that the focal adhesion-targeted, phosphatidylinositol 4,5-biphosphate-synthesizing enzyme PIPKI␥i2 coordinates with the pro-oncogenic molecule, Src, and the cytoskeletal adaptor molecule, talin, to regulate oncogenic growth of tumor cells. This is consistent with results showing that PIPKI␥ expression correlates with poor breast cancer patient survival (25) and supports a potential role for PIPKI␥ and PIPKI␥i2 in tumor progression.
The activation of Src is a hallmark of many tumors, and several mechanisms are reported for Src activation (29,30). Inactive Src largely remains in the perinuclear region, whereas FIGURE 8. Highly conserved basic amino acids residues in the N terminus of Src are required for oncogenic signaling in synergy with PIPKI␥i2. A, schematic diagram depicting the highly conserved basic amino acid residues in N terminus of Src. Mutant forms of the chicken Src used for the study are indicated. N-term., N-terminal; C-term., C-terminal. B, HEK293 cells were co-transfected with PIPKI␥i2 and Src or its mutant forms. Cells were harvested 24 hours post-transfection to immunoprecipitate PIPKI␥i2. Co-immunoprecipitation of Src was examined by immunoblotting. C, Src or its mutant forms were expressed either individually or with PIPKI␥i2 into NIH3T3 cells using lentivirus. 24 -48 h post-infection, cells were harvested and cultured in soft agar for 10 -12 days followed by counting of the colonies formed. All the values are means Ϯ S.D. from three-independent experiments. The error bars represent S.D. (p values are indicated). NS, nonsignificant p value. D, schematic diagram depicting the collaborative function of PIPKI␥i2, Src, and talin in oncogenic growth. PIPKI␥i2 simultaneously binds with Src and talin independent of cell adhesion. PIPKI␥i2 interaction with Src at its C terminus may help to alleviate intramolecular constraints, promoting Src activation. In turn, this enhances PIPKI␥i2 interaction with talin, which promotes PIPKI␥i2 and Src to localize at the proximity of integrin and growth factor receptors promoting oncogenic growth signaling.
active Src is targeted to the plasma membrane/cell adhesion sites in an actin-dependent manner (30). Myristoylation is required for Src recruitment to the plasma membrane and its activation (31). In addition, highly conserved basic residues at the N terminus of Src play an important role in Src function and its recruitment to the plasma membrane via electrostatic interaction with anionic phospholipids (32). Phosphoinositides, including phosphatidylinositol 4,5-biphosphate, constitute the pivotal lipid molecules in the plasma membrane that play key roles in the recruitment of signaling molecules possessing phosphoinositide-binding motifs and/or domains (43). The inability of Src mutants (substitution of basic residues to neutral) to interact with and function in synergy with PIPKI␥i2 to induce anchorage-independent growth strongly suggests the phosphoinositide regulation of Src function and functional integration of Src into phosphoinositide signaling pathways. Binding data indicate that PIPKI␥i2 interacts with the C terminus of Src. This interaction may relieve the intramolecular interaction between the C-tail and Src homology 2 domain, facilitating Src activation. However, PIPKI␥i2 interaction with Src C terminus did not abrogate the Src association with Csk nor Csk phosphorylation of Src. In the absence of PIPKI␥/ PIPKI␥i2 expression or in cells expressing low levels of PIPKI␥, Src may remain in an inactive state. PIPKI␥/PIPKI␥i2 might be playing a role in Src activation as well as its targeting to plasma membrane/focal adhesion sites in coordination with talin. This is consistent with the results that show a requirement for PIPKI␥i2 and talin in localization of Src at focal adhesions in adherent conditions and at cell-cell contact sites in three-dimensional culture.
Talin, Src, and PIPKI␥i2, are all targeted to focal adhesions in adherent cells. In the absence of cell-matrix interactions in suspension culture, all of them are assembled into a complex, presumably at the vicinity of integrins and growth factor receptors that sustain oncogenic signaling necessary for anchorage-independent growth. In three-dimensional culture, their mutual interaction may promote their organization at cell-cell contact sites in the plasma membrane, although their major fractions remain in cytosol (22,44,45). Furthermore, Src is required for PIPKI␥i2 association with talin in different tumor cells (not shown), and Src expression significantly promoted PIPKI␥i2 association with talin. Conversely, talin interaction with actin is regulated by phosphatidylinositol 4,5-biphosphate (46), suggesting their integrative and collaborative function. As talin is emerging as a potential regulator of oncogenesis, Src regulation of PIPKI␥i2 interaction with talin is fully consistent with a collaborative role in anchorage-independent growth (5,47,48).
In the plasma membrane, PIPKI␥i2 and activated Src may induce oncogenic signaling that contributes to anchorage-independent growth. Talin, a cytoskeletal protein and phosphatidylinositol 4,5-biphosphate effector protein, is an important component of this signaling nexus because it may selectively promote the PIPKI␥i2 targeting to the plasma membrane in suspension culture because of its ability to interact with actin cytoskeleton. With these results, we have uncovered the mechanism of how focal adhesion molecules PIPKI␥i2, Src, and talin converge into a signaling complex to support oncogenic growth of tumor cells. This could be an oncogenic axis required for in vivo tumor growth and metastasis, including that of triple negative breast cancers, where PIPKI␥ and Src are predominantly overexpressed (23,49). Furthermore, the elucidation of oncogenic signaling molecules downstream of PIPKI␥i2 and Src and their functional relevance in vivo are future directions for study.