Characterization of cortactin as an in vivo protein kinase D substrate: interdependence of sites and potentiation by Src.
Protein Kinase D (PKD) has been implicated in the regulation of actin turnover at the leading edge, invasion and migration. In particular, a complex between cortactin, paxillin and PKD in the invadopodia of invasive breast cancer cells has been described earlier, but so far this complex remained ill defined. Here we have investigated the possible role of PKD as a cortactin kinase. Using a mass spectrometric approach, we found that PKD phosphorylates cortactin on Ser 298 in the 6th cortactin repeat region and on Ser 348, right before the helical-proline rich domain of cortactin. We developed phosphospecific antibodies against these phosphorylated sequences, and used them as tools to follow the in vivo phosphorylation of cortactin by PKD. Examination of cortactin phosphorylation kinetics revealed that Ser 298 serves as a priming site for subsequent phosphorylation of Ser 348. Src, a well-known cortactin kinase, strongly potentiated the in vivo PKD mediated cortactin phosphorylation. This Src effect is neither mediated by pre-phosphorylation of cortactin nor by activation of PKD by Src. Phosphorylation of cortactin by PKD does not affect its subcellular localization, nor does it affect its translocation to podosomes or membrane ruffles. Moreover, there was no effect of PKD mediated cortactin phosphorylation on EGF receptor degradation and LPA induced migration. Taken together, these data establish cortactin as a novel PKD substrate and reveal a novel connection between Src and PKD.
De Kimpe
Line
L
Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Belgium.
Janssens
Katrien
K
Derua
Rita
R
Armacki
Milena
M
Goicoechea
Silvia
S
Otey
Carol
C
Waelkens
Etienne
E
Vandoninck
Sandy
S
Vandenheede
Jackie R
JR
Seufferlein
Thomas
T
Van Lint
Johan
J
D000595:Amino Acid Sequence; D002460:Cell Line; D051356:Cortactin / Q000276:immunology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*
D051356:Cortactin; C089212:protein kinase D; D016392:Proto-Oncogene Proteins pp60(c-src); D011493:Protein Kinase C