Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck.
BACKGROUND
The adaptor protein Nck consists of three Src homology 3 (SH3) domains followed by one SH2 domain. Like the Grb2 adaptor protein, which is known to couple receptor tyrosine kinases to the small GTPase Ras, Nck is presumed to bind to tyrosine-phosphorylated proteins using its SH2 domain and to downstream effector proteins using its SH3 domain. Little is known, however, about the specific biological function of Nck. The Pak family of serine/threonine kinases are known to be activated by binding to the GTP-bound form of Cdc42 or Rac1, which are small GTPases of the Rho family that are involved in regulating the organization of the actin cytoskeleton.


RESULTS
We present evidence that Nck can mediate the relocalization and subsequent activation of the Pak1 kinases. We show that Nck associates in vivo with Pak using the second of its three SH3 domains, and that localization of this individual Nck SH3 domain, or of Pak kinase itself, to the membrane results in activation of Pak and stimulation of downstream mitogen activated protein kinase cascades. Activation of downstream signaling by the membrane-localized Nck SH3 domain is blocked by a kinase-inactive mutant form of Pak1.


CONCLUSIONS
These results demonstrate that localization of Pak1 to the membrane in the absence of other signals is sufficient for its activation, and imply that the Nck adaptor protein could function to link changes in tyrosine phosphorylation of cellular proteins to the Cdc42/Pak signaling pathway.
Lu
W
W
Howard Hughes Medical Institute, Children's Hospital, Boston, Massachusetts 02115, USA.
Katz
S
S
Gupta
R
R
Mayer
B J
BJ
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002460:Cell Line; D004625:Embryo, Nonmammalian / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005775:Gastrula / Q000502:physiology*; D006801:Humans; D008954:Models, Biological; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D014981:Xenopus / Q000196:embryology; D018631:ras Proteins / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C078332:Nck protein; D015513:Oncogene Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D020558:GTP Phosphohydrolases; D018631:ras Proteins