Ying Hao Chou

Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin

As cells enter mitosis, the intermediate filament (IF) networks of interphase BHK-21 cells are depolymerized to form cytoplasmic aggregates of disassembled IFs, and the constituent IF proteins, vimentin and desmin are hyperphosphorylated at several specific sites. We have characterized one of two endogenous vimentin kinases from a particulate fraction of mitotic cell lysates. Through several purification steps, vimentin kinase activity copurifies with histone H1 kinase and both activities bind to p13suc1-Sepharose. The final enriched kinase preparation consists primarily of p34cdc2 and polypeptides of 65 and 110 kd. The purified kinase complex phosphorylates vimentin in vitro at a subset of sites phosphorylated in vivo during mitosis. Furthermore, phosphorylation of in vitro polymerized vimentin IFs by the purified kinase causes their disassembly. Therefore, vimentin is a substrate of p34cdc2 and phosphorylation of vimentin contributes to M phase reorganization of the IF network.

Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments

Intermediate filaments (IFs) continuously exchange between a small, depolymerized fraction of IF protein and fully polymerized IFs. To elucidate the possible role of phosphorylation in regulating this equilibrium, we disrupted the exchange of phosphate groups by specific inhibition of dephosphorylation and by specific phosphorylation and site-directed mutagenesis of two of the major in vivo phosphorylation sites determined in this study. Inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases in BHK-21 fibroblasts with calyculin-A, induced rapid vimentin phosphorylation in concert with disassembly of the IF polymers into soluble tetrameric vimentin oligomers. This oligomeric composition corresponded to the oligopeptides released by cAMP-dependent kinase (PKA) following in vitro phosphorylation. Characterization of the 32P-labeled vimentin phosphopeptides, demonstrated Ser-4, Ser-6, Ser-7, Ser-8, Ser-9, Ser-38, Ser-41, Ser-71, Ser-72, Ser-418, Ser-429, Thr-456, and Ser-457 as significant in vivo phosphorylation sites. A number of the interphase-specific high turnover sites were shown to be in vitro phosphorylation sites for PKA and protein kinase C (PKC). The effect of presence or absence of phosphate groups on individual subunits was followed in vivo by microinjecting PKA-phosphorylated (primarily S38 and S72) and mutant vimentin (S38:A, S72:A), respectively. The PKA-phosphorylated vimentin showed a clearly decelerated filament formation in vivo, whereas obstruction of phosphorylation at these sites by site-directed mutagenesis had no significant effect on the incorporation rates of subunits into assembled polymers. Taken together, our results suggest that elevated phosphorylation regulates IF assembly in vivo by changing the equilibrium constant of subunit exchange towards a higher off-rate.

A High Molecular Weight Intermediate Filament-associated Protein in BHK-21 Cells Is Nestin, a Type VI Intermediate Filament Protein LIMITED CO-ASSEMBLY IN VITRO TO FORM HETEROPOLYMERS WITH TYPE III VIMENTIN AND TYPE IV α-INTERNEXIN

BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein α-internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells.

A nestin scaffold links Cdk5/p35 signaling to oxidant‐induced cell death

The intermediate filament protein, nestin, has been implicated as an organizer of survival‐determining signaling molecules. When nestin expression was related to the sensitivity of neural progenitor cells to oxidant‐induced apoptosis, nestin displayed a distinct cytoprotective effect. Oxidative stress in neuronal precursor cells led to downregulation of nestin with subsequent activation of cyclin‐dependent kinase 5 (Cdk5), a crucial kinase in the nervous system. Nestin downregulation was a prerequisite for the Cdk5‐dependent apoptosis, as overexpression of nestin efficiently inhibited induction of apoptosis, whereas depletion of nestin by RNA interference had a sensitizing effect. When the underlying link between nestin and Cdk5 was analyzed, we observed that nestin serves as a scaffold for Cdk5, with binding restricted to a specific region following the alpha‐helical domain of nestin, and that the presence and organization of nestin regulated the sequestration and activity of Cdk5, as well as the ubiquitylation and turnover of its regulator, p35. Our data imply that nestin is a survival determinant whose action is based upon a novel mode of Cdk5 regulation, affecting the targeting, activity, and turnover of the Cdk5/p35 signaling complex.

Keratin 8 Phosphorylation by Protein Kinase C δ Regulates Shear Stress-mediated Disassembly of Keratin Intermediate Filaments in Alveolar Epithelial Cells

Phosphorylation of keratin intermediate filaments (IF) is known to affect their assembly state and organization; however, little is known about the mechanisms regulating keratin phosphorylation. In this study, we demonstrate that shear stress, but not stretch, causes disassembly of keratin IF in lung alveolar epithelial cells (AEC) and that this disassembly is regulated by protein kinase C δ-mediated phosphorylation of keratin 8 (K8) Ser-73. Specifically, in AEC subjected to shear stress, keratin IF are disassembled, as reflected by their increased solubility. In contrast, AEC subjected to stretch showed no changes in the state of assembly of IF. Pretreatment with the protein kinase C (PKC) inhibitor, bisindolymaleimide, prevents the increase in solubility of either K8 or its assembly partner K18 in shear-stressed AEC. Phosphoserine-specific antibodies demonstrate that K8 Ser-73 is phosphorylated in a time-dependent manner in shear-stressed AEC. Furthermore, we showed that shear stress activates PKC δ and that the PKC δ peptide antagonist, δ V1-1, significantly attenuates the shear stress-induced increase in keratin phosphorylation and solubility. These data suggested that shear stress mediates the phosphorylation of serine residues in K8, leading to the disassembly of IF in alveolar epithelial cells. Importantly, these data provided clues regarding a molecular link between mechanically induced signal transduction and alterations in cytoskeletal IF.

Tracking melanosomes inside a cell to study molecular motors and their interaction

Cells known as melanophores contain melanosomes, which are membrane organelles filled with melanin, a dark, nonfluorescent pigment. Melanophores aggregate or disperse their melanosomes when the host needs to change its color in response to the environment (e.g., camouflage or social interactions). Melanosome transport in cultured Xenopus melanophores is mediated by myosin V, heterotrimeric kinesin-2, and cytoplasmic dynein. Here, we describe a technique for tracking individual motors of each type, both individually and in their interaction, with high spatial (≈2 nm) and temporal (≈1 msec) localization accuracy. This method enabled us to observe (i) stepwise movement of kinesin-2 with an average step size of 8 nm; (ii) smoother melanosome transport (with fewer pauses), in the absence of intermediate filaments (IFs); and (iii) motors of actin filaments and microtubules working on the same cargo nearly simultaneously, indicating that a diffusive step is not needed between the two systems of transport. In concert with our previous report, our results also show that dynein-driven retrograde movement occurs in 8-nm steps. Furthermore, previous studies have shown that melanosomes carried by myosin V move 35 nm in a stepwise fashion in which the step rise-times can be as long as 80 msec. We observed 35-nm myosin V steps in melanophores containing no IFs. We find that myosin V steps occur faster in the absence of IFs, indicating that the IF network physically hinders organelle transport.

Intermediate filaments: not so tough after all

The dynamic properties of cellular protein polymers such as microtubules and microfilaments depend to a large extent on the cells capacity to modify rapidly the exchange rate between polymerized and unpolymerized pools of subunits. Until quite recently the dynamic nature of intermediate filaments was underestimated because of their biochemical stability in vitro and a paucity of studies on their characteristics in vivo. However, the recent studies described in this review show that the karyoskeletal and cytoskeletal structures that assemble from many intermediate filament proteins possess the properties expected of dynamic protein polymer networks.

The dynamic properties of intermediate filaments during organelle transport.

Intermediate filament (IF) dynamics during organelle transport and their role in organelle movement were studied using Xenopus laevis melanophores. In these cells, pigment granules (melanosomes) move along microtubules and microfilaments, toward and away from the cell periphery in response to α-melanocyte stimulating hormone (α-MSH) and melatonin, respectively. In this study we show that melanophores possess a complex network of vimentin IFs which interact with melanosomes. IFs form an intricate, honeycomb-like network that form cages surrounding individual and small clusters of melanosomes, both when they are aggregated and dispersed. Purified melanosome preparations contain a substantial amount of vimentin, suggesting that melanosomes bind to IFs. Analyses of individual melanosome movements in cells with disrupted IF networks show increased movement of granules in both anterograde and retrograde directions, further supporting the notion of a melanosome-IF interaction. Live imaging reveals that IFs, in turn, become highly flexible as melanosomes disperse in response to α-MSH. During the height of dispersion there is a marked increase in the rate of fluorescence recovery after photobleaching of GFP-vimentin IFs and an increase in vimentin solubility. These results reveal a dynamic interaction between membrane bound pigment granules and IFs and suggest a role for IFs as modulators of granule movement.

Structural changes in intermediate filament networks alter the activity of insulin-degrading enzyme

The intermediate filament (IF) protein nestin coassembles with vimentin and promotes the disassembly of these copolymers when vimentin is hyperphosphorylated during mitosis. The aim of this study is to determine the function of these nonfilamentous particles by identifying their interacting partners. In this study, we report that these disassembled vimentin/nestin complexes interact with insulin degrading enzyme (IDE). Both vimentin and nestin interact with IDE in vitro, but vimentin binds IDE with a higher affinity than nestin. Although the interaction between vimentin and IDE is enhanced by vimentin phosphorylation at Ser‐55, the interaction between nestin and IDE is phosphorylation independent. Further analyses show that phosphorylated vimentin plays the dominant role in targeting IDE to the vimentin/nestin particles in vivo, while the requirement for nestin is related to its ability to promote vimentin IF disassembly. The binding of IDE to either nestin or phosphorylated vimentin regulates IDE activity differently, depending on the substrate. The insulin degradation activity of IDE is suppressed ̃50% by either nestin or phosphorylated vimentin, while the cleavage of bradykinin‐mimetic peptide by IDE is increased 2‐ to 3‐fold. Taken together, our data demonstrate that the nestin‐mediated disassembly of vimentin IFs generates a structure capable of sequestering and modulating the activity of IDE.—Chou, Y.‐H., Kuo, W.‐L., Rich Rosner, M., Tang, W.‐J., Goldman, R. D. Structural changes in intermediate filament networks alter the activity of insulin‐degrading enzyme. FASEB J. 23, 3734–3742 (2009). www.fasebj.org

Dynamic aspects of cytoskeletal and karyoskeletal intermediate filament systems during the cell cycle

IF are major cytoskeletal and karyoskeletal components of eukaryotic cells (Steinert and Roop 1988). In numerous instances, their constituent protein subunits have been shown to be substrates for a variety of kinases such as A-kinase, C-kinase, and Ca++/calmodulin kinase (Geisler and Weber 1988; Inagaki et. 1988; Ando et al. 1991), as well as p34cdc2 (Chou et al. 1990; Peter et al. 1990; Ward and Kirschner 1990; Dessev et al. 1991). To date, all of the phosphorylation sites that have been mapped are in the non-alpha-helical amino- or carboxy-terminal domains (Steinert 1988; Ando et al. 1989, 1991; Geisler et al. 1989; Chou et al. 1991), and these secondary modifications can lead to IF reorganization and/or disassembly in vivo and in vitro (see, e.g., Iganaki et al. 1988; Lamb et al. 1989; Chou et al. 1990; Peter et al. 1990; Heald and McKeon 1990; Dessev et al. 1991). In addition, it is possible that the exchange seen between subunits and polymerized IF in interphase following the microinjection of unpolymerized protein (Vikstrom et al. 1989; Miller et al. 1991) may also be regulated in some fashion by phosphorylation/dephosphorylation reactions. In cultured fibroblasts such as BHK-21, the interphase equilibrium state that favors IF polymerization is shifted dramatically to a disassembled state in mitosis, apparently due to enhanced phosphorylation at specific sites mediated through the activity of p34cdc2. However, in other cells in mitosis, such as HeLa, the mechanisms involved in the regulation of cytoskeletal IF remain unclear. Therefore, no one common mechanism appears to be responsible for IF regulation during cell division. On the basis of the majority of data available, it appears that the regulation of IF phosphorylation plays an important role in the regulation of the supramolecular organization of IF cytoskeletal and karyoskeletal networks, especially in the remodeling events that take place as cells enter and exit mitosis. Although the functional significance of IF phosphorylation during interphase is not as obvious as it is in some mitotic cells, we are tempted to speculate that there may be a connection with mechanisms involved in signal transduction, since IF proteins appear to be targets for kinases known to be activated by second messengers such as Ca++ and cAMP.