Protein kinase Cα promotes cell migration through a PDZ-dependent interaction with its novel substrate discs large homolog 1 (DLG1).
Protein scaffolds maintain precision in kinase signaling by coordinating kinases with components of specific signaling pathways. Such spatial segregation is particularly important in allowing specificity of signaling mediated by the 10-member family of protein kinase C (PKC) isozymes. Here we identified a novel interaction between PKCα and the Discs large homolog (DLG) family of scaffolds that is mediated by a class I C-terminal PDZ (PSD-95, disheveled, and ZO1) ligand unique to this PKC isozyme. Specifically, use of a proteomic array containing 96 purified PDZ domains identified the third PDZ domains of DLG1/SAP97 and DLG4/PSD95 as interaction partners for the PDZ binding motif of PKCα. Co-immunoprecipitation experiments verified that PKCα and DLG1 interact in cells by a mechanism dependent on an intact PDZ ligand. Functional assays revealed that the interaction of PKCα with DLG1 promotes wound healing; scratch assays using cells depleted of PKCα and/or DLG1 have impaired cellular migration that is no longer sensitive to PKC inhibition, and the ability of exogenous PKCα to rescue cellular migration is dependent on the presence of its PDZ ligand. Furthermore, we identified Thr-656 as a novel phosphorylation site in the SH3-Hook region of DLG1 that acts as a marker for PKCα activity at this scaffold. Increased phosphorylation of Thr-656 is correlated with increased invasiveness in non-small cell lung cancer lines from the NCI-60, consistent with this phosphorylation site serving as a marker of PKCα-mediated invasion. Taken together, these data establish the requirement of scaffolding to DLG1 for PKCα to promote cellular migration.
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093, USA.
Audrey K
AK
O'Neill
Lisa L
LL
Gallegos
Verline
V
Justilien
Erin L
EL
Garcia
Michael
M
Leitges
Alan P
AP
Fields
Randy A
RA
Hall
Alexandra C
AC
Newton
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D000074288:Discs Large Homolog 1 Protein; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D054731:PDZ Domains / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering
D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D000074288:Discs Large Homolog 1 Protein; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D051571:Protein Kinase C-alpha