Gisela Link

Protein kinase D regulates vesicular transport by phosphorylating and activating phosphatidylinositol-4 kinase IIIβ at the Golgi complex

Protein kinase D (PKD) regulates the fission of vesicles originating from the trans-Golgi network. We show that phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) — a key player in the structure and function of the Golgi complex — is a physiological substrate of PKD. Of the three PKD isoforms, only PKD1 and PKD2 phosphorylated PI4KIIIβ at a motif that is highly conserved from yeast to humans. PKD-mediated phosphorylation stimulated lipid kinase activity of PI4KIIIβ and enhanced vesicular stomatitis virus G-protein transport to the plasma membrane. The identification of PI4KIIIβ as one of the PKD substrates should help to reveal the molecular events that enable transport-carrier formation.

Protein Kinase D Regulates Cell Migration by Direct Phosphorylation of the Cofilin Phosphatase Slingshot 1 Like

Protein kinase D (PKD) has been identified as a negative regulator of epithelial cell migration; however, its molecular substrates and downstream signaling pathways that mediate this activity have remained elusive. In this study, we provide evidence that the cofilin phosphatase slingshot 1 like (SSH1L), an important regulator of the complex actin remodeling machinery, is a novel in vivo PKD substrate. PKD-mediated phosphorylation of serines 937 and 978 regulates SSH1L subcellular localization by binding of 14-3-3 proteins and thus impacts the control of local cofilin activation and actin remodeling during cell migration. In line with this, we show that the loss of PKD decreases cofilin phosphorylation, induces a more spread cell morphology, and stimulates chemotactic migration of breast cancer cells in an SSHL1-dependent fashion. Our data thus identify PKD as a central regulator of the cofilin signaling network via direct phosphorylation and regulation of SSH1L.

Structural requirements for localization and activation of protein kinase C μ (PKCμ) at the Golgi compartment

We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC)μ involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKCμ–green fluorescent protein fusion proteins costained with the Golgi compartment–specific markers p24 and p230. Deletions of either the NH2-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKCμ completely abrogated Golgi localization of PKCμ. As an NH2-terminal PKCμ fragment was colocalized with p24, this region of PKCμ is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKCμ to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinase-dead PKCμ found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKCμ, in which Golgi compartment recruitment precedes and is essential for activation loop phoshorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of NH2-terminal serine(s) in the regulatory domain. PKCμ activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKCμ function at the Golgi compartment.

In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase Cμ

To study enzymatic activity and activation conditions of the recently identified novel protein kinase C μ (PKCμ) subtype, epitope tagged PKCμ was propagated in the baculovirus expression system and was purified to homogeneity. PKCμ displays high affinity phorbol ester binding (K d = 7 nM) resulting in enhanced phosphatidylserine‐dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns‐4,5‐P2, were found to promote PKCμ kinase activity. Two peptides derived from the glycogen synthase, GS‐peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine‐rich C‐kinase substrate) served as an in vitro substrate for PKCμ too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKCμ.

Phospho-specific binding of 14-3-3 proteins to phosphatidylinositol 4-kinase III β protects from dephosphorylation and stabilizes lipid kinase activity

Phosphatidylinositol-4-kinase-IIIβ (PI4KIIIβ) is activated at the Golgi compartment by PKD-mediated phosphorylation. Subsequent mechanisms responsible for continuous PtdIns(4)P production at Golgi membranes and potential interaction partners of activated PI4KIIIβ are unknown. Here we identify phosphoserine/-threonine binding 14-3-3 proteins as novel regulators of PI4KIIIβ activity downstream of this phosphorylation. The PI4KIIIβ-14-3-3 interaction, evident from GST pulldowns, co-immunoprecipitations and bimolecular fluorescence complementation, was augmented by phosphatase inhibition with okadaic acid. Binding of 14-3-3 proteins to PI4KIIIβ involved the PKD phosphorylation site Ser294, evident from reduced 14-3-3 binding to a S294A PI4KIIIβ mutant. Expression of dominant negative 14-3-3 proteins resulted in decreased PI4KIIIβ Ser294 phosphorylation, whereas wildtype 14-3-3 proteins increased phospho-PI4KIIIβ levels. This was because of protection of PI4KIIIβ Ser294 phosphorylation from phosphatase-mediated dephosphorylation. The functional significance of the PI4KIIIβ-14-3-3 interaction was evident from a reduction of PI4KIIIβ activity upon dominant negative 14-3-3 protein expression. We propose that 14-3-3 proteins function as positive regulators of PI4KIIIβ activity by protecting the lipid kinase from active site dephosphorylation, thereby ensuring a continuous supply of PtdIns(4)P at the Golgi compartment.

Protein kinase C μ selectively activates the mitogen-activated protein kinase (MAPK) p42 pathway

Here we show that human protein kinase C μ (PKCμ) activates the mitogen‐activated protein kinase (MAPK). Transient expression of constitutive active PKCμ leads to an activation of Raf‐1 kinase as demonstrated by in vitro phosphorylation of MAPK. PKCμ enhances transcriptional activity of a basal thymidine kinase promotor containing serum response elements (SREs) as shown by luciferase reporter gene assays. SRE driven gene activation by PKCμ is triggered by the Elk‐1 ternary complex factor. PKCμ‐mediated activation of SRE driven transcription can be inhibited by the MEK1 inhibitor PD98059. In contrast to the activation of the p42/ERK1 MAPK cascade, transient expression of constitutive active PKCμ does neither affect c‐jun N‐terminal kinase nor p38 MAPK.

Bruton’s tyrosine kinase (Btk) associates with protein kinase C μ

Brutons tyrosine kinase (Btk) is considered an essential signal transducer in B‐cells. Mutational defects are associated with a severe immunodeficiency syndrome, X‐chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCμ, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B‐cells with phorbol ester or H2O2 affected Btk/PKCμ interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCμ. Transient overexpression of PKCμ deletion mutants as well as expression of selected PKCμ domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH‐TH domain. These data show the existence of a PKCμ/Btk complex in vivo and identify two PKCμ domains that participate in Btk interaction.

A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration

RIN1 is a regulator of epithelial cell migration. We identify serine 292 as a novel phosphorylation site for PKD in RIN1. Phosphorylation at this site controls RIN1-mediated inhibition of cell migration by modulating the direct activation of Abl kinases.

A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal.

The protein kinase D (PKD) family comprises multifunctional serine/threonine‐specific protein kinases with three mammalian isoforms: PKD1, PKD2 and PKD3. A prominent PKD function is the regulation of basolateral‐targeted transport carrier fission from the trans‐Golgi network (TGN). To visualize site‐specific PKD activation at this organelle, we designed a molecular reporter consisting of a PKD‐specific substrate sequence fused to enhanced green fluorescent protein (EGFP), specifically targeted to the TGN via the p230 GRIP domain. Quantitative analyses using a phosphospecific antibody and ratiometric fluorescence imaging revealed that Golgi‐specific phosphorylation of the reporter was strictly dependent on stimulation of endogenous PKD or transient expression of active PKD constructs. Conversely, PKD‐specific pharmacological inhibitors and siRNA‐mediated PKD knockdown suppressed reporter phosphorylation. Using this reporter we investigated a potential role for PKD in the regulation of Golgi complex morphology. Interestingly, nocodazole‐induced Golgi complex break‐up and dispersal was associated with local PKD activation as measured by reporter phosphorylation and this was efficiently blocked by expression of a dominant‐negative PKD mutant or PKD depletion. Our data thus identify a novel link between PKD activity and the microtubule cytoskeleton, whereby Golgi complex integrity is regulated.

Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1

By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity.