ABSTRACT
The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.
Subject(s)
Cell Polarity/physiology , Drosophila Proteins/metabolism , Eukaryotic Cells/enzymology , Protein Kinase C/metabolism , Proteins/metabolism , Tumor Suppressor Proteins , Amino Acid Sequence/physiology , Animals , Brain/enzymology , COS Cells , Drosophila Proteins/genetics , Humans , Macromolecular Substances , Mice , Multiprotein Complexes , Phosphorylation , Protein Binding/physiology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase C/genetics , Protein Structure, Tertiary/physiology , Protein Transport/physiology , Proteins/genetics , Tumor Cells, Cultured , Wound Healing/physiologyABSTRACT
WAVE-1, which is also known as Scar, is a scaffolding protein that directs actin reorganization by relaying signals from the GTPase Rac to the Arp2/3 complex. Although the molecular details of WAVE activation by Rac have been described, the mechanisms by which these signals are terminated remain unknown. Here we have used tandem mass spectrometry to identify previously unknown components of the WAVE signalling network including WRP, a Rac-selective GTPase-activating protein. WRP binds directly to WAVE-1 through its Src homology domain 3 and specifically inhibits Rac function in vivo. Thus, we propose that WRP is a binding partner of WAVE-1 that functions as a signal termination factor for Rac.
Subject(s)
Microfilament Proteins/physiology , rac GTP-Binding Proteins/physiology , Amino Acid Sequence , Animals , Binding Sites , Molecular Sequence Data , Protein Binding , Rats , Rats, Sprague-Dawley , Sequence Alignment , Wiskott-Aldrich Syndrome Protein FamilyABSTRACT
Signal peptide peptidase (SPP) catalyzes intramembrane proteolysis of some signal peptides after they have been cleaved from a preprotein. In humans, SPP activity is required to generate signal sequence-derived human lymphocyte antigen-E epitopes that are recognized by the immune system, and to process hepatitis C virus core protein. We have identified human SPP as a polytopic membrane protein with sequence motifs characteristic of the presenilin-type aspartic proteases. SPP and potential eukaryotic homologs may represent another family of aspartic proteases that promote intramembrane proteolysis to release biologically important peptides.
