Role of the Second Cysteine-rich Domain and Pro275 in Protein Kinase D2 Interaction with ADP-Ribosylation Factor 1,Trans-Golgi Network Recruitment, and Protein Transport

Abstract

The present study provides the first link between the “classical” machinery regulating protein transport at the Golgi compartment, namely ARF proteins and PKDs, and demonstrates that the direct interaction of both is crucial for efficient protein transport from the TGN to the plasma membrane.

Protein Kinase D Regulates RhoA Activity via Rhotekin Phosphorylation

“…1A). In addition, phosphorylation of rhotekin was examined in the presence of PKD2 wild type (WT), kinase-dead (DA), and constitutively active (SSEE) mutants (27). As shown in Fig.…”

“…DNA Constructs-GFP-PKD1, EGFP-PKD2 WT, EGFP-PKD2 D695A (DA), EGFP-PKD2 S706E/S710E (SSEE), and GFP-PKD3 have been described previously (27). pRK5-FLAGrhotekin was a kind gift from Prof. K. Nagata (Kasugai, Japan).…”

“…Immunoprecipitation and Western Blotting-Immunoprecipitations and Western blotting were performed as described previously (27). Briefly, transfected HEK-293T cells were lysed in radioimmunoprecipitation assay lysis buffer (50 mM TrisHCl, pH 8.8, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 2.5 mM MgCl 2 , protease and phosphatase inhibitor mixture (Roche Applied Science)).…”

“…In Vitro Kinase Assay-An in vitro kinase assay was performed as described previously (27). Briefly, to examine the in vitro rhotekin phosphorylation by PKDs and their mutants, HEK-293T cells expressing GFP-tagged PKDs or PKD2 mutants were left either stimulated (ϩ) or unstimulated (Ϫ) with PMA (400 nM, 10 min) and lysed in lysis buffer I (50 mM Tris-HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol (DTT), 1% Triton X-100, and protease and phosphatase inhibitor mixture).…”

The phosphatase of regenerating liver 3 (PRL-3) promotes cell migration via Arf-activity dependent stimulation of integrin alpha5 recycling

“…To generate the GST-COPZ1, COPZ1 coding sequence was inserted into the pGEX-6P-1. The GSTArf1 construct has been described previously (Pusapati et al, 2010). Arf1-mRuby was generated by PCR, using the GST-Arf1 as a template with a forward primer (59-GCGGTACCATGGGGAACATCTTCGCC-39), and a reverse primer (59-GCCTCGAGCTTCTGGTTCCGGAGCTG-39).…”