Dematin interacts with the Ras-guanine nucleotide exchange factor Ras-GRF2 and modulates mitogen-activated protein kinase pathways.
Erythroid dematin is a major component of red blood cell junctional complexes that link the spectrin-actin cytoskeleton to the overlying plasma membrane. Transcripts of dematin are widely distributed including human brain, heart, lung, skeletal muscle, and kidney. In vitro, dematin binds and bundles actin filaments in a phosphorylation-dependent manner. The primary structure of dematin consists of a C-terminal domain homologous to the 'headpiece' domain of villin, an actin-binding protein of the brush border cytoskeleton. Except filamentous actin, no other binding partners of dematin have been identified. To investigate the physiological function of dematin, we employed the yeast two-hybrid assay to identify dematin-interacting proteins in the adult human brain. Here, we show that dematin interacts with the guanine nucleotide exchange factor Ras-GRF2 by yeast two-hybrid assay, and this interaction is further confirmed by blot overlay, surface plasmon resonance, co-transfection, and co-immunoprecipitation assays. Human Ras-GRF2 is expressed in a variety of tissues and, similar to other guanine nucleotide exchange factors (GEFs), displays anchorage independent growth in soft agar. Co-transfection and immunoblotting experiments revealed that dematin blocks transcriptional activation of Jun by Ras-GRF2 and activates ERK1 via a Ras-GRF2 independent pathway. Because much of the present evidence has centered on the identification of the Rho family of GTPases as key regulators of the actin cytoskeleton, the direct association between dematin and Ras-GRF2 may provide an alternate mechanism for regulating the activation of Rac and Ras GTPases via the actin cytoskeleton.
Lutchman
Mohini
M
Section of Hematology-Oncology Research, Department of Medicine, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135, USA.
Kim
Anthony C
AC
Cheng
Li
L
Whitehead
Ian P
IP
Oh
S Steven
SS
Hanspal
Manjit
M
Boukharov
Andrey A
AA
Hanada
Toshihiko
T
Chishti
Athar H
AH
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000378:metabolism* / Q000502:physiology; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D004847:Epithelial Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D010750:Phosphoproteins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques; D020663:ras Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001798:Blood Proteins; D003598:Cytoskeletal Proteins; C044805:DMTN protein, human; D017931:DNA Primers; C489041:Dmtn protein, mouse; D008840:Microfilament Proteins; D010750:Phosphoproteins; C504490:RASGRF2 protein, human; C504491:Rasgrf2 protein, mouse; D011994:Recombinant Proteins; D020663:ras Guanine Nucleotide Exchange Factors