Protein kinase D regulates membrane trafficking and sorting of dendritic proteins in hippocampal neurons.

BISBAL M; DONOSO M; BOLLATI F; SESMA J; QUIROGA S; DIAZ AÑEL A; MALHOTRA V; MARZOLO M; CACERES A

Villa Carlos Paz

Workshop; International Workshop on Membrane Trafficking.; 2005

Protein kinase D1 (PKD1), also known as protein kinase Cµ (PKCµ), is a member of a novel family of dyacylglycerol (DAG)-stimulated Ser/Thr kinases [1, 2]. Isoforms of PKD1 comprise, PKD2 [1] and PKD3 [1]. All these enzymes share a similar modular structure, with their catalytic domain displaying very low homology to the conserved kinase domain of all other PKC family members [2-3]. PKDs can be activated by different stimuli through a PKC-dependent pathway [1, 4-5]. Accordingly, PKDs have been implicated in diverse cellular functions, including regulation of the ERK and JNK pathways [6-8], cell survival [9, 10], apoptosis [11], tumor cell invasion [12], and immune responses [13].
Recent studies have also implicated PKD1-3 in the regulation of Golgi organization [7, 14] and plasma membrane directed transport [14-16]. Thus, PKD1-3 binds primarily to the trans-
Golgi network (TGN) through its first cysteine-rich domain in a DAG-dependent manner [14, 17]. Suppression of PKD1-3 activity inhibits a membrane fission pathway specifically involved in the transport of cargo carrying basolateral sorting signals in MDCK cells [18]. Overexpression of PKD kinase-defective mutants also leads to extensive tubulation of the TGN, with cargo-containing vesicles failing to detach from the TGN [15, 19]. Conversely, overexpression of PKD1 over activates the fission reaction leading to Golgi fragmentation, and event inhibited by expression of a kinase-defective mutant of PKD1 [19].
Neurons are highly polarized cells typically extending several short, thick tapering dendrites and one functional distinct long thin axon. Consistent with their different functions, many cell membrane proteins are preferentially distributed either to axons or dendrites, and neurons use different and complementary mechanisms to achieve this goal. It is now accepted that each step along the neuronal membrane trafficking pathway -sorting into carrier vesicles, fission and exit from the Golgi, transport along microtubules, fusion with the plasma membrane, and retention at the plasma membrane- is a potential ?decision site? where molecular selectivity mechanisms could act to govern protein targeting [20, 21]. The identification of proteins (or motifs and/or sorting signals) and/or signaling pathways that mediate and/or control these ?steps? is therefore of key importance for understanding neuronal polarity. In this regard, the possible involvement of PKD1-3 could be of particular interest.
Therefore, in this study we have analyzed the involvement of PKD1 in the sorting of dendritic and axonal membrane proteins. Specifically, our results show that reducing PKD1 proteins levels and/or activity decrease the trafficking and membrane insertion of the dendritic membrane protein mLRP4 (Low-density receptor-related protein 4), but not of the axonal membrane protein L1. After PKD1 inactivation, mLRP4 distributed to axons and dendrites but was preferentially deliver to the axonal membrane; this behavior, which is completely different from that observed in neurons with normal levels/activity of PKD1, is identical to that of L1.