Focal adhesion kinase facilitates platelet-derived growth factor-BB-stimulated ERK2 activation required for chemotaxis migration of vascular smooth muscle cells

The focal adhesion (FAK) non-receptor protein-tyrosine kinase (PTK) links both extracellular matrix/integrin and growth factor stimulation to intracellular signals promoting cell migration. Here we show that both transient and stable overexpression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) inhibits serum and platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (SMC) migration in wound healing and in vitro Boyden Chamber chemotaxis assays, respectively. Expression of FRNK, but not a point mutant of FRNK (FRNK L1034S), disrupted the formation of a complex containing both FAK and the activated PDGF-beta receptor and resulted in reduced tyrosine phosphorylation of endogenous FAK at the Tyr-397 binding site for Src family PTKs. As demonstrated using FAK-deficient and FAK-reconstituted fibroblasts, FAK positively contributed to PDGF-BB-stimulated ERK2/MAP kinase activity, and in SMCs, ERK2/MAP kinase activity was required for PDGF-BB-stimulated chemotaxis. Stable expression of FRNK but not FRNK L1034S expression in SMCs lowered the extent and duration of stimulated ERK2/MAP kinase activation at low but not at high PDGF-BB concentrations. Importantly, stable expression of FRNK in SMCs did not affect SMC morphology or proliferation in culture. Because the increased migration of vascular SMCs in response to extracellular matrix proteins and growth factors contributes to neointima formation, our results show that FAK inhibition by FRNK expression may provide a novel approach to regulate abnormal vascular SMC migration in vivo.     

Focal adhesion kinase: in command and control of cell motility

A central question in cell biology is how membrane-spanning receptors transmit extracellular signals inside cells to modulate cell adhesion and motility. Focal adhesion kinase (FAK) is a crucial signalling component that is activated by numerous stimuli and functions as a biosensor or integrator to control cell motility. Through multifaceted and diverse molecular connections, FAK can influence the cytoskeleton, structures of cell adhesion sites and membrane protrusions to regulate cell movement.     

Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.     

Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells

Focal adhesion kinase-null (FAK(-/-) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK(-/-) cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK(-/-) cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a beta 1-integrin-containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK(-/-) cells, exhibit elevated FN-stimulated extracellular signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH(2)-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to beta-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.     

Integrin α4 Enhances Metastasis and May Be Associated with Poor Prognosis in MYCNlow Neuroblastoma

High-risk neuroblastoma is associated with an overall survival rate of 30–50%. Neuroblastoma-expressed cell adhesion receptors of the integrin family impact cell adhesion, migration, proliferation and survival. Integrin α4 is essential for neural crest cell motility during development, is highly expressed on leukocytes, and is critical for transendothelial migration. Thus, cancer cells that express this receptor may exhibit increased metastatic potential. We show that α4 expression in human and murine neuroblastoma cell lines selectively enhances in vitro interaction with the alternatively spliced connecting segment 1 of fibronectin, as well as vascular cell adhesion molecule-1 and increases migration. Integrin α4 expression enhanced experimental metastasis in a syngeneic tumor model, reconstituting a pattern of organ involvement similar to that seen in patients. Accordingly, antagonism of integrin α4 blocked metastasis, suggesting adhesive function of the integrin is required. However, adhesive function was not sufficient, as mutants of integrin α4 that conserved the matrix-adhesive and promigratory function in vitro were compromised in their metastatic capacity in vivo. Clinically, integrin α4 is more frequently expressed in non-MYNC amplified tumors, and is selectively associated with poor prognosis in this subset of disease. These results reveal an unexpected role for integrin α4 in neuroblastoma dissemination and identify α4 as a potential prognostic indicator and therapeutic target.     

Growth Rates and Body Condition in Norops polylepis (Polychrotidae) Vary with Respect to Sex but not Mite Load1

Norops polylepis is a common anoline lizard that occupies both forest and pasture habitats at various elevations in southern Costa Rica. Previous studies have documented significant spatial variation in mite loads (Eutrombicula chiggers) on N. polylepis. My objective was to investigate whether mite load has a negative impact on growth rates and body condition. Growth rates were estimated in three sites using mark‐recapture with three intensive sampling periods over 12 mo. Nine hundred and forty lizards were captured and 200 individuals were recaptured across sampling periods. Mite load was significantly higher in forests than in pastures, and varied significantly among study sites. Males grew faster than females, and females and juveniles had higher body condition than males. I was unable, however, to detect any influence of mite load on growth rates or body condition. I conclude that Eutrombicula chiggers are either largely benign for N. polylepis or, if they do exert a negative effect on fitness, it is mediated through a trait other than growth or body condition.     

Cortactin as a Target for FAK in the Regulation of Focal Adhesion Dynamics

Background

Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.

Principal Findings

Using FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin.

Conclusions

Our results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement.     

Compensatory role for Pyk2 during angiogenesis in adult mice lacking endothelial cell FAK

Focal adhesion kinase (FAK) plays a critical role during vascular development because knockout of FAK in endothelial cells (ECs) is embryonic lethal. Surprisingly, tamoxifen-inducible conditional knockout of FAK in adult blood vessels (inducible EC-specific FAK knockout [i-EC-FAK-KO]) produces no vascular phenotype, and these animals are capable of developing a robust growth factor-induced angiogenic response. Although angiogenesis in wild-type mice is suppressed by pharmacological inhibition of FAK, i-EC-FAK-KO mice are refractory to this treatment, which suggests that adult i-EC-FAK-KO mice develop a compensatory mechanism to bypass the requirement for FAK. Indeed, expression of the FAK-related proline-rich tyrosine kinase 2 (Pyk2) is elevated and phosphorylated in i-EC-FAK-KO blood vessels. In cultured ECs, FAK knockdown leads to increased Pyk2 expression and, surprisingly, FAK kinase inhibition leads to increased Pyk2 phosphorylation. Pyk2 can functionally compensate for the loss of FAK because knockdown or pharmacological inhibition of Pyk2 disrupts angiogenesis in i-EC-FAK-KO mice. These studies reveal the adaptive capacity of ECs to switch to Pyk2-dependent signaling after deletion or kinase inhibition of FAK.