EGFR transactivates RON to drive oncogenic crosstalk

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation – arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.

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EGFR transactivates RON to drive oncogenic crosstalk

10.1101/2020.08.11.246785 ◽

2020 ◽

Author(s):

Carolina Franco Nitta ◽

Ellen W. Green ◽

Elton D. Jhamba ◽

Justine M. Keth ◽

Iraís Ortiz-Caraveo ◽

...

Keyword(s):

Receptor Tyrosine Kinase ◽

Structural Features ◽

Single Particle Tracking ◽

Membrane Receptors ◽

Therapeutic Resistance ◽

High Resolution Imaging ◽

Oncogenic Signaling ◽

Resolution Imaging ◽

Stimulation Of ◽

Formation Of Complexes

SUMMARYCrosstalk between disparate membrane receptors is thought to drive oncogenic signaling and allow for therapeutic resistance. EGFR and RON are members of two unique receptor tyrosine kinase (RTK) subfamilies that engage in crosstalk through unknown mechanisms. We combined high resolution imaging with biochemical studies and structural mutants to understand how EGFR and RON communicate. We found that EGF stimulation results in EGFR-dependent RON phosphorylation. Crosstalk is unidirectional, since MSP stimulation of RON does not trigger EGFR phosphorylation. Two-color single particle tracking captured the formation of complexes between RON and EGFR, supporting a role for direct interactions in propagating crosstalk. We further show that RON is a substrate for EGFR kinase, and transactivation of RON requires the formation of a signaling competent EGFR dimer. These results identify critical structural features of EGFR/RON crosstalk and provide new mechanistic insights into therapeutic resistance.

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CD44 Selectively Associates With Active Src Family Protein Tyrosine Kinases Lck and Fyn in Glycosphingolipid-Rich Plasma Membrane Domains of Human Peripheral Blood Lymphocytes

Blood ◽

10.1182/blood.v91.10.3901.3901_3901_3908 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3901-3908 ◽

Cited By ~ 3

Author(s):

Subburaj Ilangumaran ◽

Anne Briol ◽

Daniel C. Hoessli

Keyword(s):

Plasma Membrane ◽

T Lymphocytes ◽

Peripheral Blood ◽

Tyrosine Kinases ◽

Human Peripheral Blood ◽

Membrane Microdomains ◽

Low Density ◽

Membrane Domains ◽

Protein Tyrosine ◽

Plasma Membrane Microdomains

CD44 is the major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is implicated in a variety of biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of signal transduction via CD44 remains undefined, although CD44 was shown to physically associate with intracellular protein tyrosine kinase Lck in T lymphocytes. In the present report, we show that a significant proportion of CD44 in human peripheral blood T lymphocytes and endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions. CD44-associated protein kinase activity was selectively recovered from the low-density membrane fractions, corresponding to glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of the in vitro phosphorylated proteins showed that CD44 associates not only with Lck but also with Fyn kinase in these membrane domains. Our results suggest that cellular stimulation via CD44 may proceed through the signaling machinery of glycosphingolipid-enriched plasma membrane microdomains and, hence, depend on the functional integrity of such domains.

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The role of cholesterol and sphigomyelin in tyrosine phosphorylation of proteins and capping of Fcgamma receptor II.

Acta Biochimica Polonica ◽

10.18388/abp.1999_4188 ◽

1999 ◽

Vol 46 (1) ◽

pp. 107-116 ◽

Cited By ~ 7

Author(s):

A Drzewiecka ◽

K Kwiatkowska ◽

A Sobota

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Membrane Microdomains ◽

Cross Linking ◽

Fcgamma Receptor ◽

Surface Receptors ◽

Phosphorylation Of Proteins ◽

Beta Cyclodextrin ◽

Plasma Membrane Microdomains

Cross-linking of cell surface receptors by multivalent ligands, e.g. by antibodies, evokes their clustering -- patching. Subsequently, these clusters can be translocated by the acto-myosin machinery toward one pole of the cell and assembly cap. Patching of FcgammaRII in U937 cells correlates with tyrosine phosphorylation of several proteins while cap assembly correlates with their dephosphorylation. To study the mechanism of activation of tyrosine kinases during FcgammaRII activation we disturbed the organization of the putative plasma membrane microdomains by depletion of membrane cholesterol and sphingomyelin. Cholesterol was removed with the use of beta-cyclodextrin while sphingomyelin was decomposed by exogenous sphingomyelinase. Cyclodextrin at 5-10 mM removed about 70% of cholesterol from the cells and abolished the assembly of FcgammaRII caps thereby arresting the receptors at the patching stage. Similarly, 70 mU/ml sphingomyelinase inhibited cap formation by 60%. Cholesterol and sphingomyelin depletion also suppressed the tyrosine phosphorylation of proteins which accompanied cross-linking of FcgammaRII. The observations indicate that cholesterol and sphingomyelin can control the interactions of tyrosine kinases with clustered FcgammaRII.

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CD44 Selectively Associates With Active Src Family Protein Tyrosine Kinases Lck and Fyn in Glycosphingolipid-Rich Plasma Membrane Domains of Human Peripheral Blood Lymphocytes

Blood ◽

10.1182/blood.v91.10.3901 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3901-3908 ◽

Cited By ~ 98

Author(s):

Subburaj Ilangumaran ◽

Anne Briol ◽

Daniel C. Hoessli

Keyword(s):

Plasma Membrane ◽

T Lymphocytes ◽

Peripheral Blood ◽

Tyrosine Kinases ◽

Human Peripheral Blood ◽

Membrane Microdomains ◽

Low Density ◽

Membrane Domains ◽

Protein Tyrosine ◽

Plasma Membrane Microdomains

Abstract CD44 is the major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is implicated in a variety of biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of signal transduction via CD44 remains undefined, although CD44 was shown to physically associate with intracellular protein tyrosine kinase Lck in T lymphocytes. In the present report, we show that a significant proportion of CD44 in human peripheral blood T lymphocytes and endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions. CD44-associated protein kinase activity was selectively recovered from the low-density membrane fractions, corresponding to glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of the in vitro phosphorylated proteins showed that CD44 associates not only with Lck but also with Fyn kinase in these membrane domains. Our results suggest that cellular stimulation via CD44 may proceed through the signaling machinery of glycosphingolipid-enriched plasma membrane microdomains and, hence, depend on the functional integrity of such domains.

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Association and activation of Btk and Tec tyrosine kinases by gp130, a signal transducer of the interleukin-6 family of cytokines

Blood ◽

10.1182/blood.v85.3.627.bloodjournal853627 ◽

1995 ◽

Vol 85 (3) ◽

pp. 627-633 ◽

Cited By ~ 108

Author(s):

T Matsuda ◽

M Takahashi-Tezuka ◽

T Fukada ◽

Y Okuyama ◽

Y Fujitani ◽

...

Keyword(s):

Tyrosine Kinase ◽

Interleukin 6 ◽

Tyrosine Kinases ◽

Catalytic Domain ◽

Oncostatin M ◽

B Lymphopoiesis ◽

Tec Kinases ◽

Ligand Stimulation ◽

Stimulating Factor ◽

Stimulation Of

Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotrophic factor constitute the IL-6 family of cytokines and play important roles in hematopoiesis, immune response, and nervous system. The receptors for the IL-6 family of cytokines share gp130 through which signals are generated, although the cytoplasmic region of gp130 does not contain any catalytic domain. In this study we show that in addition to Jak family tyrosine kinase, the stimulation of gp130 by IL-6 plus soluble IL-6 receptor alpha induced the activation of Btk and Tec tyrosine kinases, whereas IL-3 and granulocyte colony-stimulating factor activated Tec but not Btk in a pro-B cell line. Furthermore, both Btk and Tec kinases were associated with gp130 without the ligand stimulation. Because Btk is a critical tyrosine kinase for B lymphopoiesis and Tec is considered to be involved in hematopoiesis, the results suggest the involvement of gp130-Btk-Tec signal pathway in early lymphohematopoiesis.

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Faculty Opinions recommendation of Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1026541.328582 ◽

2005 ◽

Author(s):

Ken Jacobson

Keyword(s):

T Cells ◽

Plasma Membrane ◽

Single Molecule ◽

Signaling Molecules ◽

Membrane Microdomains ◽

Protein Networks ◽

Single Molecule Microscopy ◽

Plasma Membrane Microdomains

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TAM Receptor Inhibition–Implications for Cancer and the Immune System

Cancers ◽

10.3390/cancers13061195 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1195

Author(s):

Pia Aehnlich ◽

Richard Morgan Powell ◽

Marlies J. W. Peeters ◽

Anne Rahbech ◽

Per thor Straten

Keyword(s):

T Cells ◽

Immune Responses ◽

Cancer Cells ◽

Immune Cells ◽

Tyrosine Kinases ◽

Apoptotic Cell ◽

Receptor Activation ◽

Mouse Tumor ◽

Oncogenic Signaling ◽

Tam Receptors

Tyro3, Axl and MerTK (TAM) receptors are receptor tyrosine kinases which play important roles in efferocytosis and in the balancing of immune responses and inflammation. TAM receptor activation is induced upon binding of the ligands protein S (Pros1) or growth arrest-specific protein 6 (Gas6) which act as bridging molecules for binding of phosphatidyl serine (PtdSer) exposed on apoptotic cell membranes. Upon clearance of apoptotic cell material, TAM receptor activation on innate cells suppresses proinflammatory functions, thereby ensuring the immunologically silent removal of apoptotic material in the absence of deleterious immune responses. However, in T cells, MerTK signaling is costimulatory and promotes activation and functional output of the cell. MerTK and Axl are also aberrantly expressed in a range of both hematological and solid tumor malignancies, including breast, lung, melanoma and acute myeloid leukemia, where they have a role in oncogenic signaling. Consequently, TAM receptors are being investigated as therapeutic targets using small molecule inhibitors and have already demonstrated efficacy in mouse tumor models. Thus, inhibition of TAM signaling in cancer cells could have therapeutic value but given the opposing roles of TAM signaling in innate cells and T cells, TAM inhibition could also jeopardize anticancer immune responses. This conflict is discussed in this review, describing the effects of TAM inhibition on cancer cells as well as immune cells, while also examining the intricate interplay of cancer and immune cells in the tumor microenvironment.

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Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment

The Journal of Cell Biology ◽

10.1083/jcb.201005140 ◽

2010 ◽

Vol 191 (4) ◽

pp. 771-781 ◽

Cited By ~ 76

Author(s):

Alexander Ludwig ◽

Grant P. Otto ◽

Kirsi Riento ◽

Emily Hams ◽

Padraic G. Fallon ◽

...

Keyword(s):

Plasma Membrane ◽

Ex Vivo ◽

Neutrophil Migration ◽

Neutrophil Recruitment ◽

Membrane Microdomains ◽

Neutrophil Chemotaxis ◽

Myosin Iia ◽

Plasma Membrane Microdomains ◽

Cortical Cytoskeleton

We studied the function of plasma membrane microdomains defined by the proteins flotillin 1 and flotillin 2 in uropod formation and neutrophil chemotaxis. Flotillins become concentrated in the uropod of neutrophils after exposure to chemoattractants such as N-formyl-Met-Leu-Phe (fMLP). Here, we show that mice lacking flotillin 1 do not have flotillin microdomains, and that recruitment of neutrophils toward fMLP in vivo is reduced in these mice. Ex vivo, migration of neutrophils through a resistive matrix is reduced in the absence of flotillin microdomains, but the machinery required for sensing chemoattractant functions normally. Flotillin microdomains specifically associate with myosin IIa, and spectrins. Both uropod formation and myosin IIa activity are compromised in flotillin 1 knockout neutrophils. We conclude that the association between flotillin microdomains and cortical cytoskeleton has important functions during neutrophil migration, in uropod formation, and in the regulation of myosin IIa.

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