Donald J. Fujita

Activating SRC mutation in a subset of advanced human colon cancers.

The discovery of Rous sarcoma virus (RSV) led to the identification of cellular Src (c–Src), a non-receptor tyrosine kinase, which has since been implicated in the development of numerous human cancers. c-Src has been found to be highly activated in colon cancers, particularly in those metastatic to the liver. Studies of the mechanism of c-Src regulation have suggested that c-Src kinase activity is downregulated by phosphorylation of a critical carboxy-terminal tyrosine (Tyr 530 in human c-Src, equivalent to Tyr 527 in chicken Src) and have implied the existence of activating mutations in this C-terminal regulatory region. We report here the identification of a truncating mutation in SRC at codon 531 in 12% of cases of advanced human colon cancer tested and demonstrate that the mutation is activating, transforming, tumorigenic and promotes metastasis. These results provide, for the first time, genetic evidence that activating SRC mutations may have a role in the malignant progression of human colon cancer.

Selected glimpses into the activation and function of Src kinase

Since the discovery of the v-src and c-src genes and their products, much progress has been made in the elucidation of the structure, regulation, localization, and function of the Src protein. Src is a non-receptor protein tyrosine kinase that transduces signals that are involved in the control of a variety of cellular processes such as proliferation, differentiation, motility, and adhesion. Src is normally maintained in an inactive state, but can be activated transiently during cellular events such as mitosis, or constitutively by abnormal events such as mutation (i.e. v-Src and some human cancers). Activation of Src occurs as a result of disruption of the negative regulatory processes that normally suppress Src activity, and understanding the various mechanisms behind Src activation has been a target of intense study. Src associates with cellular membranes, in particular the plasma membrane, and endosomal membranes. Studies indicate that the different subcellular localizations of Src could be important for the regulation of specific cellular processes such as mitogenesis, cytoskeletal organization, and/or membrane trafficking. This review will discuss the history behind the discovery and initial characterization of Src and the regulatory mechanisms of Src activation, in particular, regulation by modification of the carboxy-terminal regulatory tyrosine by phosphatases and kinases. Its focus will then turn to the different subcellular localizations of Src and the possible roles of nuclear and perinuclear targets of Src. Finally, a brief section will review some of our present knowledge regarding Src involvement in human cancers.

Identification of Protein-tyrosine Phosphatase 1B as the Major Tyrosine Phosphatase Activity Capable of Dephosphorylating and Activating c-Src in Several Human Breast Cancer Cell Lines

c-Src tyrosine kinase activity is elevated in several types of human cancer, and this has been attributed to elevated c-Src expression levels, increased c-Src specific activity, and activating mutations in c-Src. We have found a number of human breast cancer cell lines with elevated c-Src specific activity that also possess elevated phosphatase activity directed against the carboxyl-terminal negative regulatory domain of Src family kinases. To identify this phosphatase, cell extracts from MDA-MB-435S cells were chromatographed and the fractions were assayed for phosphatase activity. Four peaks of phosphatase activity directed against the nonspecific substrate poly(Glu/Tyr) were detected. One peak also dephosphorylated a peptide modeled against the c-Src carboxyl-terminal negative regulatory domain and intact human c-Src. Immunoblotting and immunodepletion experiments identified the phosphatase as protein-tyrosine phosphatase 1B (PTP1B). Examination of several human breast cancer cell lines with increased c-Src activity showed elevated levels of PTP1B protein relative to normal control breast cells.In vitro c-Src reactivation experiments confirmed the ability of PTP1B to dephosphorylate and activate c-Src. In vivo overexpression of PTP1B in 293 cells caused a 2-fold increase of endogenous c-Src kinase activity. Our findings indicate that PTP1B is the primary protein-tyrosine phosphatase capable of dephosphorylating c-Src in several human breast cancer cell lines and suggests a regulatory role for PTP1B in the control of c-Src kinase activity.

REAP: A two minute cell fractionation method

BackgroundThe translocation or shuttling of proteins between the nucleus and cytoplasm (nucleocytoplasmic transport [NCPT]) is often a rapid event following stimulation with growth factors or in response to stress or other experimental manipulations. Commonly used methods to separate nuclei from cytoplasm employ lengthy steps such as density gradient centrifugation which exposes cells to non-physiological hyperosmotic conditions for extended time periods resulting in varying degrees of leakage between the nucleus and cytoplasm. To help maintain and quantify nuclear:cytoplasmic ratios of proteins, agents such as leptomycin B have been employed to be able to better analyze NCPT by inhibiting nuclear export. To track NCPT in the absence of these experimental manipulations that could introduce unknown artefacts, we have developed a rapid method that appears to produce pure nuclear and cytoplasmic fractions, suitable for obtaining accurate estimates of the nuclear:cytoplasmic ratios of proteins known to undergo NCPT.FindingsWe have developed a R apid, E fficient A nd P ractical (REAP) method for subcellular fractionation of primary and transformed human cells in culture. The REAP method is a two minute non-ionic detergent-based purification technique requiring only a table top centrifuge, micro-pipette and micro-centrifuge tubes. This inexpensive method has proven to efficiently separate nuclear from cytoplasmic proteins as estimated by no detectible cross-contamination of the nucleoporin and lamin A nuclear markers or the pyruvate kinase and tubulin cytoplasmic markers. REAP fractions also mirrored TNFα induced NF-κB NCPT observed in parallel by indirect immunofluorescence.ConclusionsThis method drastically reduces the time needed for subcellular fractionation, eliminates detectable protein degradation and maintains protein interactions. The simplicity, brevity and efficiency of this procedure allows for tracking ephemeral changes in subcellular relocalization of proteins while maintaining protein integrity and protein complex interactions.

A Dual Inhibitor of Platelet-Derived Growth Factor β-Receptor and Src Kinase Activity Potently Interferes With Motogenic and Mitogenic Responses to PDGF in Vascular Smooth Muscle Cells A Novel Candidate for Prevention of Vascular Remodeling

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

PTP1B Contributes to the Oncogenic Properties of Colon Cancer Cells through Src Activation

Src-specific activity has been reported to be elevated in a high percentage of colon cancer cell lines and tumors, but the underlying mechanisms are largely unknown. In this study, we report that, in the seven cancer cell lines tested, Src-specific activity was elevated (5.2- to 18.7-fold) relative to normal colon cells (FHC). This activation of Src correlated with reduced phosphorylation at Y530 of Src, whereas there was no significant change in the level of phosphorylation at Y419. The membrane tyrosine phosphatase activity for a Src family-specific phosphopeptide substrate FCP (Fyn COOH-terminal peptide phosphorylated by Csk) was greatly increased in the cancer cells and was attributed to PTP1B in most of the cell lines. Membrane PTP1B protein levels were also greatly increased. Overexpression of PTP1B increased Src specific activity in colon cancer cells by reducing phosphorylation at Y530 of Src. It also increased anchorage-independent cell growth and this increase was blocked by the Src inhibitor PP2 and Src small interfering RNA (siRNA). Down-regulating PTP1B activity by PTP1B inhibitor CinnGEL 2Me or knocking down PTP1B using siRNA also reduced Src kinase activity and colony formation ability of colon cancer cells. PTP1B siRNA reduced tumor growth in nonobese diabetic/severe combined immunodeficient mice. This study suggests that (a) PTP1B can act as an important activator of Src in colon cancer cells via dephosphorylation at Y530 of Src and (b) elevated levels of PTP1B can increase tumorigenicity of colon cancer cells by activating Src.

Activation of Src in human breast tumor cell lines: elevated levels of phosphotyrosine phosphatase activity that preferentially recognizes the Src carboxy terminal negative regulatory tyrosine 530

Elevated levels of Src kinase activity have been reported in a number of human cancers, including colon and breast cancer. We have analysed four human breast tumor cell lines that exhibit high levels of Src kinase activity, and have determined that these cell lines also exhibit a high level of a phosphotyrosine phosphatase activity that recognizes the Src carboxy-terminal P-Tyr530 negative regulatory site. Total Src kinase activity in these cell lines is elevated as much as 30-fold over activity in normal control cells and specific activity is elevated as much as 5.6-fold. When the breast tumor cells were grown in the presence of the tyrosine phosphatase inhibitor vanadate, Src kinase activity was reduced in all four breast tumor cell lines, suggesting that Src was being activated by a phosphatase which could recognize the Tyr530 negative regulatory site. In fractionated cell extracts from the breast tumor cells, we found elevated levels of a membrane associated tyrosine phosphatase activity that preferentially dephosphorylated a Src family carboxy-terminal phosphopeptide containing the regulatory tyrosine 530 site. Src was hypophosphorylated in vivo at tyrosine 530 in at least two of the tumor cell lines, further suggesting that Src was being activated by a phosphatase in these cells. In preliminary immunoprecipitation and antibody depletion experiments, we were unable to correlate the major portion of this phosphatase activity with several known phosphatases.

Src Kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells

BackgroundThe cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor) – dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1) and Flt-1 (Fms-like tyrosine kinase-1). However, to date, it has not been determined which VEGF receptor (VEGFR) is involved in binding to and activating Src kinase following VEGF stimulation of the receptors.ResultsIn this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs), and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1.ConclusionsThus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events.

The RNA binding protein Sam68 is acetylated in tumor cell lines, and its acetylation correlates with enhanced RNA binding activity

Sam68 (Src-associated in mitosis; 68 kDa) is a member of the STAR (signal transduction and activation of RNA) family of KH domain-containing RNA binding proteins. Accumulating evidence suggests that it plays an important role in cell cycle control. Tyrosine phosphorylation by Src family kinases and breast tumor kinase can negatively regulate its RNA binding activity. To date, there are no reports of a factor, such as a phosphatase, which can positively regulate Sam68 association with RNA. Acetylation is a reversible post-translational modification known to influence the activity of DNA binding proteins. However, acetylation of a cellular RNA binding protein as a mechanism for regulating its activity has not yet been reported. Here we demonstrate Sam68 to be acetylated in vivo. A screen of several human mammary epithelial cell lines revealed variations in Sam68 acetylation. Interestingly, the highest level of acetylation was found in tumorigenic breast cancer cell lines. The screen also showed a positive correlation between Sam68 acetylation and its ability to bind RNA. The acetyltransferase CBP was shown to acetylate Sam68 and enhance its binding to poly(U) RNA. These results suggest that Sam68 association with RNA substrates may be positively regulated by acetylation, and that enhanced acetylation and RNA binding activity of Sam68 may play a role in tumor cell proliferation.

Tyrosine phosphorylation of annexin II tetramer is stimulated by membrane binding

In the present article we have examined if the interaction of the Ca2+-binding protein, annexin II tetramer (AIIt) with the plasma membrane phospholipids or with the submembranous cytoskeleton, effects the accessibility of the tyrosine phosphorylation site of AIIt. In the presence of Ca2+, pp60c-src catalyzed the incorporation of 0.22 ± 0.05 mol of phosphate/mol of AIIt (mean ± S.D., n = 5). The Ca2+-dependent binding of AIIt to purified adrenal medulla plasma membrane or phosphatidylserine vesicles stimulated the pp60c-src-dependent phosphorylation of AIIt to 0.62 ± 0.04 mol of phosphate/mol of AIIt (mean ± S.D., n = 5) or 0.93 ± 0.07 mol of phosphate/mol of AIIt (mean ± S.D., n = 5), respectively. Phosphatidylserine- or phosphatidylinositol-containing vesicles but not vesicles composed of phosphatidylcholine or phosphatidylethanolamine, stimulated the phosphorylation of AIIt. In contrast, the binding of AIIt to F-actin resulted in the incorporation of only 0.04 ± 0.04 mol of phosphate/mol of AIIt (mean ± S.D., n = 5). These results suggest that the interaction of AIIt with plasma membrane and not the submembranous cytoskeleton, activates the tyrosine phosphorylation of AIIt by inducing a conformational change in the protein resulting in the enhanced exposure or accessibility of the tyrosine-phosphorylation site.