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Diffusion-limited translocation mechanism of protein kinase C isotypes¹

Institut für Pharmakologie, Freie Universität Berlin, 14195 Berlin, Germany

²Correspondence: Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67–73, 14195 Berlin, Germany. E-mail: schae{at}zedat.fu-berlin.de

SPECIFIC AIMS

We aimed to clarify the mechanisms by which protein kinases C (PKC) are trafficked to the plasma membrane. We also wanted to compare the kinetics of receptor-mediated plasma membrane translocation of fluorescent classical (PKC and ßI) and novel (PKC and ) PKC fusion proteins in transiently or stably transfected living cells.

PRINCIPAL FINDINGS

1. Kinetics of receptor-mediated plasma membrane association of classical and novel PKCs
Stimulation of a histamine H₁ receptor induced a rapid and transient plasma membrane association of PKC fusion proteins (Fig. 1A ). Mean association half-times of the yellow fluorescent protein (YFP) -fused PKC isotypes were 0.4–0.8 s for PKC and PKCßI, and 4–6 s for the novel isotypes, respectively. The association half-times determined in more than 200 single HEK cells expressing the respective PKC isotype revealed an apparent lower limit of 250 ms for classical or 800 ms for novel PKCs. The dissociation half-times were in the range of 20–45 s for each isotype. An even faster dissociation was observed in stably transfected cell lines stimulated via an endogenously expressed muscarinic acetylcholine receptor. In confluent HEK cells, 20 µM carbachol frequently induced [Ca²⁺]_i oscillations with variable frequencies and amplitudes. The PKC-YFP (Fig. 1B ) repetitively translocated during [Ca²⁺]_i oscillations with a tight coupling to the frequency of secondary [Ca²⁺]_i waves. Peak values of oscillatory [Ca²⁺]_i elevations were also sensed, resulting in gradual translocation signals. Both the rising and the falling phases of [Ca²⁺]_i pulses slightly preceded the PKC movements. PKC-YFP translocated with a delayed and monophasic kinetics that was not synchronized with [Ca²⁺]_i oscillations (Fig. 1B ).

View larger version (40K): [in this window] [in a new window]   Figure 1. Kinetics of receptor-mediated translocation of PKC isotypes. A) Time courses of histamine-induced translocation of YFP-fused PKCs transiently expressed in HEK cells. Fluorescences over the plasma membrane (Fpm) were divided by the cytosolic (Fcyt) signals and normalized. Traces of single cells are depicted as gray lines; black lines indicate means of all cells. B) Confluent, Fura-2-loaded HEK cells stably expressing PKC-YFP or PKC-YFP were stimulated with 20 µM carbachol to induce oscillatory [Ca2+]i responses. Black lines represent the [Ca2+]i, whereas translocations of the respective PKC isotype are depicted by the gray lines.

2. Classical and novel PKCs are freely diffusible in the cytosol of quiescent HEK cells
Fluorescence recovery after photobleach was applied to generate fluorescence gradients in living cells and to follow their equilibration over time by confocal laser scanning microscopy. A half-maximal equilibration of intracellular fluorescence gradients was observed within 0.8–5 s depending on the size and the geometry of cells. Half-maximal equilibration of a fluorescence gradient of PKC-YFP over a 16 µm distance was observed after 4 s. The mobility of fluorescent PKCs was further tested by photobleaching a line defined in the periphery of a cell. A 4–8 s-interval was sufficient to bleach 90–98% of the total fluorescence of the cell. Thus, the majority of PKC molecules passed the line within a few seconds. The partial recovery appeared with half-times of 0.5–3 s for each of the investigated PKC isoforms. These data exclude a tight coupling to larger particles, filaments, or even organelles.

3. Diffusion coefficients and lateral mobility of PKC fusion proteins
The lateral mobility of PKCs defines the maximal speed of diffusion-driven mechanisms. Therefore, Stokes’ radii of PKC fusion proteins were determined by gel filtration. The elution volumes of fusion proteins revealed Stokes’ radii of 45–50 Å for the YFP-fused PKC isotypes , ßI, , and . Assuming an apparent viscosity of 2–3.2 cp in the cytosol, the mean lateral displacement of single PKC molecules is 5.4–7.1 µm/s². The mean length and width of HEK cells expressing PKC fusion proteins were 27 µm and 15 µm at a midnuclear plane, respectively. These values illustrate that the diffusion speed prevents PKCs from being half-maximally redistributed earlier than 0.2 s after receptor activation. Furthermore, the data exclude different diffusion velocities as a possible reason for the faster translocation of classical PKCs.

4. Subcellular kinetics of PKC translocation
The millisecond association process of classical PKCs may result from an active transport mechanism or, if driven by diffusion, requires an exceptionally efficient collisional coupling mechanism. Confocal line scan imaging at high temporal and submicron spatial resolution was applied to distinguish between the two possible mechanisms (Fig. 2 ). A directional transport mechanism would preferentially and initially deplete the inner parts of the cell (Fig. 3A ). Alternatively, a diffusion-driven model predicts that the vicinity of the plasma membrane would be initially depleted until diffusion from the perinuclear cytosol reconstitutes an equilibrium (Fig. 3B ). Lines were defined and scanned at a midnuclear plane (Fig. 2A ). Coincident with the beginning accumulation of PKC and ßI at the plasma membrane, the adjacent cytosol became preferentially depleted (Fig. 2B ). The initial depletion adjacent to the plasma membrane did not rely on inhomogeneously distributed [Ca²⁺]_i elevations and could be mimicked by photolysis of caged Ca²⁺. For PKC-YFP, a local depletion zone was visible only after appropriate scaling, whereas the slow translocation and the low efficacy of PKC binding to the plasma membrane prevented a reliable detection of a subplasmalemmal depletion (Fig. 2B ).

View larger version (52K): [in this window] [in a new window]   Figure 2. Confocal line scan imaging of translocation initiation. Confocal laser scanning microscopy of cells coexpressing PKC-YFP fusion proteins and an H1 histamine receptor. A) Midnuclear sections of the cells with the position of the lines selected for subsequent line scan imaging are given. B) Time courses of spatio-temporally resolved PKC-YFP-concentrations during repetitive scanning of a line. The fluorescence intensities along the scanned line are visualized by applying a rainbow pseudocolor scale to the data. The ordinate represents the position along the scanned line. Cells were stimulated by adding 100 µM histamine to the bath solution as indicated by the dotted line. Arrows indicate the subplasmalemmal cytosol that is depleted immediately after stimulation. A representative experiment of 10–23 line scans with similar results is shown.

View larger version (43K): [in this window] [in a new window]   Figure 3. Schematic diagram of subcellular movements of PKCs. A) An active transport mechanism that drags PKCs toward the plasma membrane would result in a directional movement of PKCs. If a line is defined in a perinuclear cytosol, the time-dependent concentration of PKC along the line would show a minimum in the center of the cell until the equilibrium is achieved. B) Diffusion-driven movement of PKCs would be nondirectional. Only in the proximity of the plasma membrane may a highly efficient docking of PKCs result in a directional movement that initially depletes the subplasmalemmal cytosol. Therefore, confocal line scan microscopy would reveal minima just beyond the plasma membrane and a local maximum in the perinuclear cytosol that equilibrate by diffusion.

5. Collisional coupling of plasma membrane binding of PKC
Assuming a Gaussian distribution of diffusion kinetics, the receptor-induced translocation of PKC was simulated to estimate the collisional coupling efficacy of the binding process. Assuming a collisional coupling of 100%, the process is only restricted by the diffusion velocity. Indeed, a lateral movement of 6 µm/s² was required to achieve half-maximal translocation times of 500 ms. The comparison of histamine-induced PKC-YFP translocation with computer simulations revealed that a diffusion speed of 6 µm/s² and a collisional coupling efficacy of 50% give a good overlap with the experimental data. Furthermore, translocations of novel PKCs are well simulated with the same diffusion velocity, but a collisional coupling of 10%. The translocation of classical PKCs in response to photolysis of caged Ca²⁺ revealed similar values for lateral movement and collisional coupling, indicating that diffusive PKC movements rather than upstream signaling events mainly limit the time course of PKC activation.

CONCLUSIONS

The spatial and temporal patterns of signal transduction contribute to the cellular decoding of hormonal signaling. Imaging of YFP-fused classical and novel PKC isoenzymes revealed a redistribution of both PKC and PKCßI to the plasma membrane with half-times of 250–800 ms. By contrast, PKC and PKC translocated 5- to 10-fold more slowly. These association kinetics agree with those observed for PKC, but most reports on GFP-fused classical PKCs described association kinetics that are much slower or not sufficiently resolved. Thus, PKCs are fast signaling molecules and phosphorylation of PKC substrates may begin within the first second after receptor stimulation. [Ca²⁺]_i oscillations in response to low agonist concentrations were synchronously followed by repetitive translocation of PKC and PKCßI. A similar pattern has been described for PKC, another classical PKC. In cells overexpressing the novel PKCs or , oscillatory changes of [Ca²⁺]_i were not followed by repetitive PKC translocations. The concept that cellular responses to hormonal stimulation are regulated by the frequency of [Ca²⁺]_i waves rather than by peak amplitude points to distinct roles for classical and novel PKCs that either follow or withstand the [Ca²⁺]_i fluctuations. Considering the fast translocation dynamics, it might be tempting to speculate that classical PKC isoforms could be an integral part of the oscillatory mechanism, a concept that has already been implicated in the generation of sinusoidal [Ca²⁺]_i oscillations.

The inability to block PKC translocation by depolymerization of filamentous actin or tubulin demonstrated that PKCs are not trafficked along these structures. Accordingly, our photobleaching experiments indicate that PKCs are freely diffusible in the cytosol. The different translocation kinetics of classical and novel PKCs might reflect a larger particle diameter of novel PKCs. However, the similar Stokes’ radii strongly argue against this concept. The mean lateral displacement of PKC fusion proteins was 6 µm/s². Thus, a single PKC molecule collides with the plasma membrane 1–3 times per second. Half-maximal translocation times of less than 1 s demonstrate that the plasma membrane of stimulated cells readily accepts but poorly reflects incoming PKC molecules (Fig. 3B ). This behavior was confirmed by observing PKC distribution at high spatio-temporal resolution. Confocal line scan imaging of PKC isoenzymes revealed that the subplasmalemmal area was first depleted whereas PKCs in the perinuclear cytosol subsequently followed, presumably driven by diffusion along the concentration gradient. The observation of a localized depletion zone is direct evidence for a diffusion-limited binding process. Diffusion limits a variety of intracellular and intercellular processes such as oxygen transport, substrate flow, and ion fluxes. To our knowledge, the rapid redistribution of classical PKCs is the first direct observation of a diffusion-limited plasma membrane binding of a protein in living cells.

Collision-coupling efficacies of 30–50% for classical PKCs suggest that binding occurs at a plasma membrane lipid rather than a protein. Nonetheless, the isotype-selective association of PKCs with specific interacting proteins is not in contrast with our observations. However, our data indicate that initial docking of PKC molecules is accomplished by a protein–lipid interaction. The Ca²⁺ binding C2 domain may, via electrostatic interaction, orientate the binding face of classical PKC toward negatively charged phospholipids in the plasma membrane. Since this effect is lacking in novel PKC isotypes, the additional binding force may serve a mechanistic model for the superior docking efficacy of classical PKCs.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0824fje ; to cite this article, use FASEB J. (May 29, 2001) 10.1096/fj.00-0824fje

³ Present address: Institut für Pharmakologie und Toxikologie, Philipps Universität Marburg, Fachbereich Humanmedizin, Karl-von-Frisch Str. 1, 35033 Marburg, Germany.

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