Johan Van Lint

Protein Kinase D Regulates the Fission of Cell Surface Destined Transport Carriers from the Trans-Golgi Network

When a kinase inactive form of Protein Kinase D (PKD-K618N) was expressed in HeLa cells, it localized to the trans-Golgi network (TGN) and caused extensive tubulation. Cargo that was destined for the plasma membrane was found in PKD-K618N-containing tubes but the tubes did not detach from the TGN. As a result, the transfer of cargo from TGN to the plasma membrane was inhibited. We have also demonstrated the formation and subsequent detachment of cargo-containing tubes from the TGN in cells stably expressing low levels of PKD-K618N. Our results suggest that PKD regulates the fission from the TGN of transport carriers that are en route to the cell surface.

Gβγ-Mediated Regulation of Golgi Organization Is through the Direct Activation of Protein Kinase D

Abstract We have shown previously that the βγ subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gβγ is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gβγ directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gβγ-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gβγ with PKD regulates the dynamics of Golgi membranes and protein secretion.

Glycogen Synthase Kinase-3β Phosphorylates Protein Tau and Rescues the Axonopathy in the Central Nervous System of Human Four-repeat Tau Transgenic Mice

Protein tau filaments in brain of patients suffering from Alzheimers disease, frontotemporal dementia, and other tauopathies consist of protein tau that is hyperphosphorylated. The responsible kinases operating in vivo in neurons still need to be identified. Here we demonstrate that glycogen synthase kinase-3β (GSK-3β) is an effective kinase for protein tau in cerebral neurons in vivo in adult GSK-3β and GSK-3β × human tau40 transgenic mice. Phosphorylated protein tau migrates slower during electrophoretic separation and is revealed by phosphorylation-dependent anti-tau antibodies in Western blot analysis. In addition, its capacity to bind to re-assembled paclitaxel (Taxol®)-stabilized microtubules is reduced, compared with protein tau isolated from mice not overexpressing GSK-3β. Co-expression of GSK-3β reduces the number of axonal dilations and alleviates the motoric impairment that was typical for single htau40 transgenic animals (Spittaels, K., Van den Haute, C., Van Dorpe, J., Bruynseels, K., Vandezande, K., Laenen, I., Geerts, H., Mercken, M., Sciot, R., Van Lommel, A., Loos, R., and Van Leuven, F. (1999) Am. J. Pathol. 155, 2153–2165). Although more hyperphosphorylated protein tau is available, neither an increase in insoluble protein tau aggregates nor the presence of paired helical filaments or tangles was observed. These findings could have therapeutic implications in the field of neurodegeneration, as discussed.

Protein kinase D: an intracellular traffic regulator on the move

Recent research has identified protein kinase D (PKD, also called PKCmu) as a serine/threonine kinase with potentially important roles in growth factor signaling as well as in stress-induced signaling. Moreover, PKD has emerged as an important regulator of plasma membrane enzymes and receptors, in some cases mediating cross-talk between different signaling systems. The recent discovery of two additional kinases belonging to the PKD family and the plethora of proteins that interact with PKD point to a multifaceted regulation and a multifunctional role for these enzymes, with functions in processes as diverse as cell proliferation, apoptosis, immune cell regulation, tumor cell invasion and regulation of Golgi vesicle fission.

Protein kinase D: a family affair.

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCμ, PKD2 and PKD3/PKCν. They all share a similar architecture with regulatory sub‐domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.

Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor–induced Nur77 expression and apoptosis

The molecular basis of thymocyte negative selection, a crucial mechanism in establishing central tolerance, is not yet resolved. Histone deacetylases (HDACs) have emerged as key transcriptional regulators in several major developmental programs. Recently, we showed that the class IIa member, HDAC7, regulates negative selection by repressing expression of Nur77, an orphan nuclear receptor involved in antigen-induced apoptosis of thymocytes. Engagement of the T cell receptor (TCR) alleviates this repression through phosphorylation-dependent nuclear exclusion of HDAC7. However, the identity of the TCR-activated kinase that phosphorylates and inactivates HDAC7 was still unknown. Here, we demonstrate that TCR-induced nuclear export of HDAC7 and Nur77 expression is mediated by activation of protein kinase D (PKD). Indeed, active PKD stimulates HDAC7 nuclear export and Nur77 expression. In contrast, inhibition of PKD prevents TCR-mediated nuclear exclusion of HDAC7 and associated Nur77 activation. Furthermore, we show that HDAC7 is an interaction partner and a substrate for PKD. We identify four serine residues in the NH2 terminus of HDAC7 as targets for PKD. More importantly, a mutant of HDAC7 specifically deficient in phosphorylation by PKD, inhibits TCR-mediated apoptosis of T cell hybridomas. These findings indicate that PKD is likely to play a key role in the signaling pathways controlling negative selection.

Recruitment of protein kinase D to the trans‐Golgi network via the first cysteine‐rich domain

Protein kinase D (PKD) is a cytosolic protein, which upon binding to the trans‐Golgi network (TGN) regulates the fission of transport carriers specifically destined to the cell surface. We have found that the first cysteine‐rich domain (C1a), but not the second cysteine‐rich domain (C1b), is sufficient for the binding of PKD to the TGN. Proline 155 in C1a is necessary for the recruitment of intact PKD to the TGN. Whereas C1a is sufficient to target a reporter protein to the TGN, mutation of serines 744/748 to alanines in the activation loop of intact PKD inhibits its localization to the TGN. Moreover, anti‐phospho‐PKD antibody, which recognizes only the activated form of PKD, recognizes the TGN‐bound PKD. Thus, activation of intact PKD is important for binding to the TGN.

Protein Kinase D Controls Actin Polymerization and Cell Motility through Phosphorylation of Cortactin

We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility.

Protein kinase D2 mediates activation of nuclear factor κB by Bcr-Abl in Bcr-Abl+ human myeloid leukemia cells

The Bcr-Abl tyrosine kinase activates various signaling pathways including nuclear factor κB that mediate proliferation, transformation, and apoptosis resistance in Bcr-Abl+ myeloid leukemia cells. Here we report that protein kinase (PK) D2, a serine threonine kinase of the PKD family, is a novel substrate of Bcr-Abl. PKD2 was found to be the major isoform of the PKD family expressed in chronic myeloid leukemia cells and is tyrosine phosphorylated by Bcr-Abl in its pleckstrin homology domain. A mutant that mimicks tyrosine phosphorylation of PKD2 in the pleckstrin homology domain activates nuclear factor κB independently of its catalytic activity. Furthermore, our data show that Bcr-Abl–induced activation of the nuclear factor κB cascade in LAMA84 cells is largely mediated by tyrosine-phosphorylated PKD2. These data present a novel mechanism of Bcr-Abl–induced nuclear factor κB activation in myeloid leukemia. Targeting PKD2 tyrosine phosphorylation, not its kinase activity, could be a novel therapeutic approach for the treatment of Bcr-Abl+ myeloid leukemia.

Protein Kinase D Induces Transcription through Direct Phosphorylation of the cAMP-response Element-binding Protein

Protein kinase D (PKD), a family of serine/threonine kinases, can be activated by a multitude of stimuli in a protein kinase C-dependent or -independent manner. PKD is involved in signal transduction pathways controlling cell proliferation, apoptosis, motility, and protein trafficking. Despite its versatile functions, few genuine in vivo substrates for PKD have been identified. In this study we demonstrate that the transcription factor cAMP-response element-binding protein (CREB) is a direct substrate for PKD. PKD1 and CREB interact in cells, and activated PKD1 provokes CREB phosphorylation at Ser-133 both in vitro and in vivo. A constitutive active mutant of PKD1 stimulates GAL4-CREB-mediated transcription in a Ser-133-dependent manner, activates CRE-responsive promoters, and increases the expression of CREB target genes. PKD1 also enhances transcription mediated by two other members of the CREB family, ATF-1 and CREM. Our results describe a novel mechanism for PKD-induced signaling through activation of the transcription factor CREB and suggest that stimulus-induced phosphorylation of CREB, reported to be mediated by protein kinase C, may involve downstream activated PKD.