Safety profiling of genetically engineered Pim-1 kinase overexpression for oncogenicity risk in human c-kit+ cardiac interstitial cells.

Advancement of stem cell-based treatment will involve next-generation approaches to enhance therapeutic efficacy which is often modest, particularly in the context of myocardial regenerative therapy. Our group has previously demonstrated the beneficial effect of genetic modification of cardiac stem cells with Pim-1 kinase overexpression to rejuvenate aged cells as well as potentiate myocardial repair. Despite these encouraging findings, concerns were raised regarding potential for oncogenic risk associated with Pim-1 kinase overexpression. Testing of Pim-1 engineered c-kit+ cardiac interstitial cells (cCIC) derived from heart failure patient samples for indices of oncogenic risk was undertaken using multiple assessments including soft agar colony formation, micronucleation, gamma-Histone 2AX foci, and transcriptome profiling. Collectively, findings demonstrate comparable phenotypic and biological properties of cCIC following Pim-1 overexpression compared with using baseline control cells with no evidence for oncogenic phenotype. Using a highly selective and continuous sensor for quantitative assessment of PIM1 kinase activity revealed a sevenfold increase in Pim-1 engineered vs. control cells. Kinase activity profiling using a panel of sensors for other kinases demonstrates elevation of IKKs), AKT/SGK, CDK1-3, p38, and ERK1/2 in addition to Pim-1 consistent with heightened kinase activity correlating with Pim-1 overexpression that may contribute to Pim-1-mediated effects. Enhancement of cellular survival, proliferation, and other beneficial properties to augment stem cell-mediated repair without oncogenic risk is a feasible, logical, and safe approach to improve efficacy and overcome current limitations inherent to cellular adoptive transfer therapeutic interventions.

An algebraic model to determine substrate kinetic parameters by global nonlinear fit of progress curves.

We propose that the time course of an enzyme reaction following the Michaelis-Menten reaction mechanism can be conveniently described by a newly derived algebraic equation, which includes the Lambert Omega function. Following Northrop's ideas [Anal. Biochem.321, 457-461, 1983], the integrated rate equation contains the Michaelis constant (KM) and the specificity number (kS≡kcat/KM) as adjustable parameters, but not the turnover number kcat. A modification of the usual global-fit approach involves a combinatorial treatment of nominal substrate concentrations being treated as fixed or alternately optimized model parameters. The newly proposed method is compared with the standard approach based on the "initial linear region" of the reaction progress curves, followed by nonlinear fit of initial rates to the hyperbolic Michaelis-Menten equation. A representative set of three chelation-enhanced fluorescence EGFR kinase substrates is used for experimental illustration. In one case, both data analysis methods (linear and nonlinear) produced identical results. However, in another test case, the standard method incorrectly reported a finite (50-70 µM) KM value, whereas the more rigorous global nonlinear fit shows that the KM is immeasurably high.

Pitfalls and Considerations in Determining the Potency and Mutant Selectivity of Covalent Epidermal Growth Factor Receptor Inhibitors.

Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure-kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The reengineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.

Structural Basis for Inhibition of Mutant EGFR with Lazertinib (YH25448).

Lazertinib (YH25448) is a novel third-generation tyrosine kinase inhibitor (TKI) developed as a treatment for EGFR mutant non-small cell lung cancer. To better understand the nature of lazertinib inhibition, we determined crystal structures of lazertinib in complex with both WT and mutant EGFR and compared its binding mode to that of structurally related EGFR TKIs. We observe that lazertinib binds EGFR with a distinctive pyrazole moiety enabling hydrogen bonds and van der Waals interactions facilitated through hydrophilic amine and hydrophobic phenyl groups, respectively. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent HER2. The molecular basis for lazertinib inhibition of EGFR reported here highlights previously unexplored binding interactions leading to improved medicinal chemistry properties compared to clinically approved osimertinib (AZD9291) and offers novel strategies for structure-guided design of tyrosine kinase inhibitors.

Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics.

Continuous adhesion formation and disassembly (adhesion turnover) in the protrusions of migrating cells is regulated by unclear mechanisms. We show that p21-activated kinase (PAK)-induced phosphorylation of serine 273 in paxillin is a critical regulator of this turnover. Paxillin-S273 phosphorylation dramatically increases migration, protrusion, and adhesion turnover by increasing paxillin-GIT1 binding and promoting the localization of a GIT1-PIX-PAK signaling module near the leading edge. Mutants that interfere with the formation of this ternary module abrogate the effects of paxillin-S273 phosphorylation. PAK-dependent paxillin-S273 phosphorylation functions in a positive-feedback loop, as active PAK, active Rac, and myosin II activity are all downstream effectors of this turnover pathway. Finally, our studies led us to identify in highly motile cells a class of small adhesions that reside near the leading edge, turnover in 20-30 s, and resemble those seen with paxillin-S273 phosphorylation. These adhesions appear to be regulated by the GIT1-PIX-PAK module near the leading edge.

Pim-1 kinase antagonizes aspects of myocardial hypertrophy and compensation to pathological pressure overload.

Pim-1 kinase exerts potent cardioprotective effects in the myocardium downstream of AKT, but the participation of Pim-1 in cardiac hypertrophy requires investigation. Cardiac-specific expression of Pim-1 (Pim-WT) or the dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice together with adenoviral-mediated overexpression of these Pim-1 constructs was used to delineate the role of Pim-1 in hypertrophy. Transgenic overexpression of Pim-1 protects mice from pressure-overload-induced hypertrophy relative to wild-type controls as evidenced by improved hemodynamic function, decreased apoptosis, increases in antihypertrophic proteins, smaller myocyte size, and inhibition of hypertrophic signaling after challenge. Similarly, Pim-1 overexpression in neonatal rat cardiomyocyte cultures inhibits hypertrophy induced by endothelin-1. On the cellular level, hearts of Pim-WT mice show enhanced incorporation of BrdU into myocytes and a hypercellular phenotype compared to wild-type controls after hypertrophic challenge. In comparison, transgenic overexpression of Pim-DN leads to dilated cardiomyopathy characterized by increased apoptosis, fibrosis, and severely depressed cardiac function. Furthermore, overexpression of Pim-DN leads to reduced contractility as evidenced by reduced Ca(2+) transient amplitude and decreased percentage of cell shortening in isolated myocytes. These data support a pivotal role for Pim-1 in modulation of hypertrophy by impacting responses on molecular, cellular, and organ levels.

Synthesis and Pharmacological Characterization of Visabron, a Backbone Cyclic Peptide Dual Antagonist of α4ß1 (VLA-4)/α9ß1 Integrin for Therapy of Multiple Sclerosis.

Integrins α4ß1/ α9ß1 are important in the pathogenesis and progression of inflammatory and autoimmune diseases by their roles in leukocyte activation and trafficking. Natalizumab, a monoclonal antibody selectively targeting α4ß1 integrin and blocking leukocyte trafficking to the central nervous system, is an immunotherapy for multiple sclerosis (MS). However, due to its adverse effects associated with chronic treatment, alternative strategies using small peptide mimetic inhibitors are being sought. In the present study, we synthesized and characterized visabron c (4-4), a backbone cyclic octapeptide based on the sequence TMLD, a non-RGD unique α4ß1 integrin recognition sequence motif derived from visabres, a proteinous disintegrin from the viper venom. Visabron c (4-4) was selected from a minilibrary with conformational diversity based on its potency and selectivity in functional adhesion cellular assays. Visabron c (4-4)'s serum stability, pharmacokinetics, and therapeutic effects following ip injection were assessed in an experimental autoimmune encephalomyelitis (EAE) animal model. Furthermore, visabron c (4-4)'s lack of toxic effects in mice was verified by blood analysis, tissue pathology, immunogenicity, and "off-target" effects, indicating its significant tolerability and lack of immunogenicity. Visabron c (4-4) can be delivered systemically. The in vitro and in vivo data justify visabron c (4-4) as a safe alternative peptidomimetic lead compound/drug to monoclonal anti-α4 integrin antibodies, steroids, and other immunosuppressant drugs. Moreover, visabron c (4-4) design may pave the way for developing new therapies for a variety of other inflammatory and/or autoimmune diseases.

Myocardial induction of nucleostemin in response to postnatal growth and pathological challenge.

Stem cell-specific proteins and regulatory pathways that determine self-renewal and differentiation have become of fundamental importance in understanding regenerative and reparative processes in the myocardium. One such regulatory protein, named nucleostemin, has been studied in the context of stem cells and several cancer cell lines, where expression is associated with proliferation and maintenance of a primitive cellular phenotype. We find nucleostemin is present in young myocardium and is also induced following cardiomyopathic injury. Nucleostemin expression in cardiomyocytes is induced by fibroblast growth factor-2 and accumulates in response to Pim-1 kinase activity. Cardiac stem cells also express nucleostemin that is diminished in response to commitment to a differentiated phenotype. Overexpression of nucleostemin in cultured cardiac stem cells increases proliferation while preserving telomere length, providing a mechanistic basis for potential actions of nucleostemin in promotion of cell survival and proliferation as seen in other cell types.

Neurotropic activity and safety of methylene-cycloalkylacetate (MCA) derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid.

Polyneuropathy is a disease involving multiple peripheral nerves injuries. Axon regrowth remains the major prerequisite for plasticity, regeneration, circuit formation, and eventually functional recovery and therefore, regulation of neurite outgrowth might be a candidate for treating polyneuropathies. In a recent study, we synthesized and established the methylene-cycloalkylacetate (MCAs) pharmacophore as a lead for the development of a neurotropic drug (inducing neurite/axonal outgrowth) using the PC12 neuronal model. In the present study we extended the characterizations of the in vitro neurotropic effect of the derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid (MCA-13) on dorsal root ganglia and spinal cord neuronal cultures and analyzed its safety properties using blood biochemistry and cell counting, acute toxicity evaluation in mice and different in vitro "off-target" pharmacological evaluations. This MCA derivative deserves further preclinical mechanistic pharmacological characterizations including therapeutic efficacy in in vivo animal models of polyneuropathies, toward development of a clinically relevant neurotropic drug.

Evolution of the c-kit-positive cell response to pathological challenge in the myocardium.

Cumulative evidence indicates that myocardium responds to growth or injury by recruitment of stem and/or progenitor cells that participate in repair and regenerative processes. Unequivocal identification of this population has been hampered by lack of reagents or markers specific to the recruited population, leading to controversies regarding the nature of these cells. Use of a transgenic mouse expressing green fluorescent protein driven by the c-kit promoter allows for unambiguous identification of this cell population. Green fluorescent protein (GFP) driven by the c-kit promoter labels a fraction of the c-kit+ cells recognized by antibody labeling for c-kit protein. Expression of GFP by the c-kit promoter and accumulation of GFP-positive cells in the myocardium is relatively high at birth compared with adult and declines between postnatal weeks 1 and 2, which tracks in parallel with expression of c-kit protein and c-kit-positive cells. Acute cardiomyopathic injury by infarction prompts increased expression of both GFP protein and GFP-labeled cells in the region of infarction relative to remote myocardium. Similar increases were observed for c-kit protein and cells with a slightly earlier onset and decline relative to the GFP signal. Cells coexpressing GFP, c-kit, and cardiogenic markers were apparent at 1-2 weeks postinfarction. Cardiac-resident c-kit+ cell cultures derived from the transgenic line express GFP that is diminished in parallel with c-kit by induction of differentiation. The use of genetically engineered mice validates and extends the concept of c-kit+ cells participating in the response to myocardial injury.

Kinetic assay for characterization of spleen tyrosine kinase activity and inhibition with recombinant kinase and crude cell lysates.

Spleen tyrosine kinase (Syk) is involved in the activation of cells implicated in allergic or autoimmune diseases and certain cancers. Therefore, Syk inhibitors may prove to be effective in treating diseases where Syk activity or expression is increased or deregulated. We developed a continuous and direct (noncoupled) fluorescence intensity assay for measuring Syk activity using purified recombinant enzyme or crude lysates generated from anti-immunoglobulin M (IgM) antibody-treated RAMOS cells. The assay is based on the chelation-enhanced fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline (referred to as Sox), which has been incorporated into a peptide substrate selected for robust detection of Syk activity. This homogeneous assay is simple to use, provides considerably more information, and has been adapted to a 384-well, low-volume microtiter plate format that can be used for the high-throughput identification and kinetic characterization of Syk inhibitors. The assay can be performed with a wide range of adenosine triphosphate (ATP) concentrations and, therefore, can be used to analyze ATP-competitive and ATP-noncompetitive/allosteric kinase inhibitors. Measurement of Syk activity in RAMOS crude cell lysates or immunoprecipitation (IP) capture formats may serve as a physiologically more relevant enzyme source. These Sox-based continuous and homogeneous assays provide a valuable set of tools for studying Syk signaling and for defining inhibitors that may be more effective in controlling disease.

Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration.

RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.

Atrial natriuretic peptide promotes cardiomyocyte survival by cGMP-dependent nuclear accumulation of zyxin and Akt.

This study delineates a mechanism for antiapoptotic signaling initiated by atrial natriuretic peptide (ANP) stimulation leading to elevation of cGMP levels and subsequent nuclear accumulation of Akt kinase associated with zyxin, a cytoskeletal LIM-domain protein. Nuclear targeting of zyxin induces resistance to cell death coincident with nuclear accumulation of activated Akt. Nuclear translocation of zyxin triggered by cGMP also promotes nuclear Akt accumulation. Additional supportive evidence for nuclear accumulation of zyxin-enhancing cardiomyocyte survival includes the following: (a) promotion of zyxin nuclear localization by cardioprotective stimuli; (b) zyxin association with phospho-Akt473 induced by cardioprotective stimuli; and (c) recruitment of zyxin to the nucleus by activated nuclear-targeted Akt as well as recruitment of Akt by nuclear-targeted zyxin. Nuclear accumulation of zyxin requires both Akt activation and nuclear localization. Potentiation of cell survival is sensitive to stimulation intensity with high-level induction by ANP or cGMP signaling leading to apoptotic cell death rather than enhancing resistance to apoptotic stimuli. Myocardial nuclear accumulation of zyxin and Akt responds similarly in vivo following treatment of mice with ANP or cGMP. Thus, zyxin and activated Akt participate in a cGMP-dependent signaling cascade leading from ANP receptors to nuclear accumulation of both molecules. Nuclear accumulation of zyxin and activated Akt may represent a fundamental mechanism that facilitates nuclear-signal transduction and potentiates cell survival.

Akt promotes increased cardiomyocyte cycling and expansion of the cardiac progenitor cell population.

Activation of Akt is associated with enhanced cell cycling and cellular proliferation in nonmyocytes, but this effect of nuclear Akt accumulation has not been explored in the context of the myocardium. Cardiac-specific expression of nuclear-targeted Akt (Akt/nuc) in transgenics prolongs postnatal cell cycling as evidenced by increased numbers of Ki67+ cardiomyocytes at 2 to 3 weeks after birth. Similarly, nuclear-targeting of Akt promotes expansion of the presumptive cardiac progenitor cell population as assessed by immunolabeling for c-kit in combination with myocyte-specific markers Nkx 2.5 or MEF 2C. Increases in pro-proliferative cytokines, including tumor-necrosis superfamily 8, interleukin-17e, and hepatocyte growth factor, were found in nuclear-targeted Akt myocardial samples. Concurrent signaling mediated by paracrine factors downstream of Akt/nuc expression may be responsible for phenotypic effects of nuclear-targeted Akt in the myocardium, including enhanced cell proliferation and expansion of the stem cell population.

Phosphorylation of phosphoprotein enriched in astrocytes (PEA-15) regulates extracellular signal-regulated kinase-dependent transcription and cell proliferation.

Cell cycle progression is dependent on the nuclear localization and transcriptional effects of activated extracellular signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinases (ERK1/2). Phosphoprotein enriched in astrocytes (PEA-15) binds ERK1/2 and inhibits their nuclear localization, thus blocking cell proliferation. Here, we report that phosphorylation of PEA-15 blocks its interaction with ERK1/2 in vitro and in vivo and that phosphorylation of both Ser104 and Ser116 is required for this effect. Using phosphomimetic and nonphosphorylatable mutants of PEA-15, we found that PEA-15 phosphorylation abrogates its capacity to block the nuclear localization and transcriptional activities of ERK1/2; this phosphorylation therefore enables the proliferation of cells that express high levels of PEA-15. Additionally, we report that PEA-15 phosphorylation can modulate nontranscriptional activities of ERK1/2, such as the modulation of the affinity of integrin adhesion receptors. Finally, we used a novel anti-phospho-specific PEA-15 antibody to establish that PEA-15 is phosphorylated in situ in normal mammary epithelium. These results define a novel posttranslational mechanism for controlling the subcellular localization of ERK1/2 and for specifying the output of MAP kinase signaling.

Phosphorylation of the Pro-X-Thr-Pro site in phosphatase inhibitor-2 by cyclin-dependent protein kinase during M-phase of the cell cycle.

Protein phosphorylation serves as a primary mechanism for triggering events during mitosis and depends on coordinated regulation of kinases and phosphatases. Protein Ser-Thr phosphatase-1 (PP1) activity is essential for the metaphase to anaphase transition and the most ancient regulator of PP1 conserved from yeast to human is inhibitor-2 (I-2), an unstructured heat-stable protein. A unique sequence motif in I-2 from various species surrounds a phosphorylation site PXTP that can be phosphorylated in biochemical assays by GSK3, MAPK and CDK kinases. Here we used a phosphosite specific antibody to investigate the phosphorylation of I-2. We fractioned extracts from HeLa cells arrested with nocodazole and assayed for PXTP kinases using recombinant I-2. One major and two minor peaks of kinase activity were identified and the major peak contained both active MAPK and cdk1::cyclinB1, confirmed by immunoblotting. Cells released from a double thymidine block synchronously progressed through mitosis and immunoblotting revealed transient phosphorylation of endogenous I-2 in cells only during mitosis, and corresponding phosphorylation of histone H3 (Ser10) and PP1 (Thr320). Activation of cdk1::cyclinB1 was coincident with I-2 phosphorylation, but neither MAPK nor GSK3 were phosphorylated at this time, so we concluded that in living cells only cdk1::cyclinB1 phosphorylated the PXTP site in I-2. Immunofluorescent staining of cells with the PXTP phosphosite antibody revealed highly specific staining of mitotic cells prior to anaphase, at which point the staining disappeared. Thus, phosphorylation of I-2 is catalyzed by cdk1::cyclinB1 and staining with a specific antibody should prove useful as a selective marker of cells in the early stages of mitosis.

The phosphorylation of vinculin on tyrosine residues 100 and 1065, mediated by SRC kinases, affects cell spreading.

Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin is tyrosine phosphorylated in platelets spread on fibrinogen and that the phosphorylation is Src kinases dependent. The phosphorylation of vinculin on tyrosine was reconstituted in vanadate treated COS-7 cells coexpressing c-Src. The tyrosine phosphorylation sites in vinculin were mapped to residues 100 and 1065. A phosphorylation-specific antibody directed against tyrosine residue 1065 reacted with phosphorylated platelet vinculin but failed to react with vinculin from unstimulated platelet lysates. Tyrosine residue 1065 located in the vinculin tail domain was phosphorylated by c-Src in vitro. When phosphorylated, the vinculin tail exhibited significantly less binding to the vinculin head domain than the unphosphorylated tail. In contrast, the phosphorylation did not affect the binding of vinculin to actin in vitro. A double vinculin mutant protein Y100F/Y1065F localized to focal adhesion plaques. Wild-type vinculin and single tyrosine phosphorylation mutant proteins Y100F and Y1065F were significantly more effective at rescuing the spreading defect of vinculin null cells than the double mutant Y100F/Y1065F. The phosphorylation of vinculin by Src kinases may be one mechanism by which these kinases regulate actin filament assembly and cell spreading.

Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton.

Cortactin is an F-actin binding protein that activates actin-related protein 2/3 complex and is localized within lamellipodia. Cortactin is a substrate for Src and other protein tyrosine kinases involved in cell motility, where its phosphorylation on tyrosines 421, 466, and 482 in the carboxy terminus is required for cell movement and metastasis. In spite of the importance of cortactin tyrosine phosphorylation in cell motility, little is known regarding the structural, spatial, or signaling requirements regulating cortactin tyrosine phosphorylation. Herein, we report that phosphorylation of cortactin tyrosine residues in the carboxy terminus requires the aminoterminal domain and Rac1-mediated localization to the cell periphery. Phosphorylation-specific antibodies directed against tyrosine 421 and 466 were produced to study the regulation and localization of tyrosine phosphorylated cortactin. Phosphorylation of cortactin tyrosine 421 and 466 was elevated in response to Src, epidermal growth factor receptor and Rac1 activation, and tyrosine 421 phosphorylated cortactin localized with F-actin in lamellipodia and podosomes. Cortactin tyrosine phosphorylation is progressive, with tyrosine 421 phosphorylation required for phosphorylation of tyrosine 466. These results indicate that cortactin tyrosine phosphorylation requires Rac1-induced cortactin targeting to cortical actin networks, where it is tyrosine phosphorylated in hierarchical manner that is closely coordinated with its ability to regulate actin dynamics.