ATP enhances neuronal differentiation of PC12 cells by activating PKCα interactions with cytoskeletal proteins.
PKCα is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its downstream signaling pathways remain to be elucidated. To identify the signaling partners of PKCα, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media. Mass spectrometry analysis (LC-MS/MS) identified plectin, peripherin, filamin A, fascin, and β-actin as potential interacting proteins. The colocalization of PKCα and its interacting proteins increased when PC12 cells were differentiated with NGF and ATP. Peripherin and plectin organization and the cortical remodeling of β-actin were dramatically affected when PKCα was down-regulated, suggesting that all three proteins might be functional targets of ATP-dependent PKCα signaling. Taken together, these data demonstrate that PKCα is essential for controlling the neuronal development induced by NGF and ATP and interacts with the cytoskeletal components at two levels: assembly of the intermediate filament peripherin and organization of cortical actin.
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Consuelo
C
Marín-Vicente
Marta
M
Guerrero-Valero
Michael L
ML
Nielsen
Mikhail M
MM
Savitski
Juan C
JC
Gómez-Fernández
Roman A
RA
Zubarev
Senena
S
Corbalán-García
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D015536:Down-Regulation; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007382:Intermediate Filaments / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D020932:Nerve Growth Factor / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016501:Neurites / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D016716:PC12 Cells; D064531:Peripherins; D051190:Plectin / Q000378:metabolism; D025941:Protein Interaction Mapping; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D051381:Rats; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000502:physiology; D053719:Tandem Mass Spectrometry
D003598:Cytoskeletal Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D064531:Peripherins; D051190:Plectin; D034741:RNA, Small Interfering; D000255:Adenosine Triphosphate; D020932:Nerve Growth Factor; D051571:Protein Kinase C-alpha