The Role of PKD in Cell Polarity, Biosynthetic Pathways, and Organelle/F-actin Distribution

Abstract

ABSTRACT. Protein Kinase D (PKD) 1, 2, and 3 are members of the PKD family. PKDs influence many cellular processes, including cell polarity, structure of the Golgi, polarized transport from the Golgi to the basolateral plasma membrane, and actin polymerization. However, the role of the PKD family in cell polarity has not yet been elucidated in vivo. Here, we show that KO mice displayed similar localization of the apical and basolateral proteins, transport of VSV-G and a GPI-anchored protein, and similar localization of actin filaments. As DKO mice were embryonic lethal, we generated MEFs that lacked all PKD isoforms from the PKD1 and PKD2 double floxed mice using Cre recombinase and PKD3 siRNA. We observed a similar localization of various organelles, a similar time course in the transport of VSV-G and a GPI-anchored protein, and a similar distribution of F-actin in the PKD-null MEFs. Collectively, our results demonstrate that the complete deletion of PKDs does not affect the transport of VSV-G or a GPI-anchored protein, and the distribution of F-actin. However, simultaneous deletion of PKD1 and PKD2 affect embryonic development, demonstrating their functional redundancy during development.

Functional redundancy of protein kinase D1 and protein kinase D2 in neuronal polarity

“…As in other tissues shown (Atik et al, 2014), PKD1 and PKD2 were absent in the brains of PKD1 geo/geo (PKD1 KO) mice and PKD2 geo/geo (PKD2 KO) mice, respectively (Fig. 1A).…”

“…Our previous work showed that PKD1 and PKD2 were expressed in various tissues, including the brain (Atik et al, 2014). As in other tissues shown (Atik et al, 2014), PKD1 and PKD2 were absent in the brains of PKD1 geo/geo (PKD1 KO) mice and PKD2 geo/geo (PKD2 KO) mice, respectively (Fig.…”

“…They were fixed by perfusion with 3% (w/v) paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4) and processed as previously described (Sato et al, 2011;Atik et al, 2014). We performed hematoxylin-eosin (HE) staining and immunofluorescence staining according to previous methods (Sato et al, 2011;Atik et al, 2014). A laser scanning confocal microscope (FV-1000D, Olympus, Tokyo, Japan) was used to analyze the samples.…”

“…Western blot analysis was performed as previously described (Sato et al, 2011;Atik et al, 2014) using 25 g of protein loaded per lane. The following primary antibodies and a reagent were used; anti-PKD1 (Sigma, St. Louis, MO, USA), anti-PKD1 (C20: Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PKD3 (Cell Signaling Technology, Danvers, MA, USA), anti-lamin B (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-tau (Tau1: Chemicon, Temecula, CA, USA), anti-GS28 (BD Biosciences, San Jose, CA, USA), anti-MAP1A (Harada et al, 1994), anti-GFP (Atik et al, 2014), and rhodamine-phalloidin (Molecular Probes, Eugene, OR, USA).…”

Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion

“…Data obtained in MEFs from mice deficient in PKD2 enzymatic activity (PKD2 S707A/S711A knock-in mutation (35,36)) further confirmed that the active kinase is indeed required for proper secretion of endogenous MMP2 cargo (Fig. 1, F and G), a function that has been also recently challenged (48). Thus, the PKD2-ARF1 protein complex is a critical dual regulator of vesicle formation and scission at the TGN during constitutive secretory processes.…”