Differential localization in cells of myosin II heavy chain kinases during cytokinesis and polarized migration

Background  

Cortical myosin-II filaments in Dictyostelium discoideum display enrichment in the posterior of the cell during cell migration and in the cleavage furrow during cytokinesis. Filament assembly in turn is regulated by phosphorylation in the tail region of the myosin heavy chain (MHC). Early studies have revealed one enzyme, MHCK-A, which participates in filament assembly control, and two other structurally related enzymes, MHCK-B and -C. In this report we evaluate the biochemical properties of MHCK-C, and using fluorescence microscopy in living cells we examine the localization of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization.     

A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly

Deletion of the synapsin I genes, encoding one of the major groups of proteins on synaptic vesicles, in mice causes late onset epileptic seizures and enhanced experimental temporal lobe epilepsy. However, mice lacking synapsin I maintain normal excitatory synaptic transmission and modulation but for an enhancement of paired-pulse facilitation. To elucidate the cellular basis for epilepsy in mutants, we examined whether the inhibitory synapses in the hippocampus from mutant mice are intact by electrophysiological and morphological means. In the cultured hippocampal synapses from mutant mice, repeated application of a hypertonic solution significantly suppressed the subsequent transmitter release, associated with an accelerated vesicle replenishing time at the inhibitory synapses, compared with the excitatory synapses. In the mutants, morphologically identifiable synaptic vesicles failed to accumulate after application of a hypertonic solution at the inhibitory preterminals but not at the excitatory preterminals. In the CA3 pyramidal cells in hippocampal slices from mutant mice, inhibitory postsynaptic currents evoked by direct electrical stimulation of the interneuron in the striatum oriens were characterized by reduced quantal content compared with those in wild type. We conclude that synapsin I contributes to the anchoring of synaptic vesicles, thereby minimizing transmitter depletion at the inhibitory synapses. This may explain, at least in part, the epileptic seizures occurring in the synapsin I mutant mice.     

Orchestrating nonmuscle myosin II filament assembly at the onset of cytokinesis

Contractile forces in the actomyosin cortex are required for cellular morphogenesis. This includes the invagination of the cell membrane during division, where filaments of nonmuscle myosin II (NMII) are responsible for generating contractile forces in the cortex. However, how NMII heterohexamers form filaments in vivo is not well understood. To quantify NMII filament assembly dynamics, we imaged the cortex of Caenorhabditis elegans embryos at high spatial resolution around the time of the first division. We show that during the assembly of the cytokinetic ring, the number of NMII filaments in the cortex increases and more NMII motors are assembled into each filament. These dynamics are influenced by two proteins in the RhoA GTPase pathway, the RhoA-dependent kinase LET-502 and the myosin phosphatase MEL-11. We find that these two proteins differentially regulate NMII activity at the anterior and at the division site. We show that the coordinated action of these regulators generates a gradient of free NMII in the cytoplasm driving a net diffusive flux of NMII motors toward the cytokinetic ring. Our work highlights how NMII filament assembly and disassembly dynamics are orchestrated over space and time to facilitate the up-regulation of cortical contractility during cytokinesis.     

Aurora-B phosphorylates the myosin II heavy chain to promote cytokinesis

Cytokinesis, the final step of mitosis, is mediated by an actomyosin contractile ring, the formation of which is temporally and spatially regulated following anaphase onset. Aurora-B is a member of the chromosomal passenger complex, which regulates various processes during mitosis; it is not understood, however, how Aurora-B is involved in cytokinesis. Here, we show that Aurora-B and myosin-IIB form a complex in vivo during telophase. Aurora-B phosphorylates the myosin-IIB rod domain at threonine 1847 (T¹⁸⁴⁷), abrogating the ability of myosin-IIB monomers to form filaments. Furthermore, phosphorylation of myosin-IIB filaments by Aurora-B also promotes filament disassembly. We show that myosin-IIB possessing a phosphomimetic mutation at T¹⁸⁴⁷ was unable to rescue cytokinesis failure caused by myosin-IIB depletion. Cells expressing a phosphoresistant mutation at T¹⁸⁴⁷ had significantly longer intercellular bridges, implying that Aurora-B-mediated phosphorylation of myosin-IIB is important for abscission. We propose that myosin-IIB is a substrate of Aurora-B and reveal a new mechanism of myosin-IIB regulation by Aurora-B in the late stages of mitosis.     

Actin-activated Mg-ATPase activity of Dictyostelium myosin II. Effects of filament formation and heavy chain phosphorylation.

The actin-activated Mg-ATPase activities of unphosphorylated and heavy chain phosphorylated Dictyostelium myosin II and of a Dictyostelium myosin II heavy meromyosin (HMM) fragment were examined at different Mg2+ and KCl concentrations. The Mg-ATPase activity of HMM displayed a maximum rate, Vmax, of about 4.0/s and a Kapp (actin concentration required to achieve 1/2 Vmax) that increased from 8 to 300 microM as the KCl concentration increased from 0 to 120 mM. When assayed with greater than 5 mM Mg2+ and 0 mM KCl the unphosphorylated Dictyostelium myosin II yielded a Kapp of 0.25 microM and a Vmax of 2.8/s. At lower Mg2+ concentrations or with 50 mM KCl the data were not fit well by a single hyperbolic curve and Kapp increased to 25-100 microM. The increase in Kapp did not correlate with the loss of sedimentable filaments. At KCl concentrations above 100 mM Vmax increased to greater than 4/s. Heavy chain phosphorylated myosin (3.5 mol of phosphate/mol myosin) displayed a Vmax of about 5/s and a Kapp of 50 microM under all conditions tested. Thus, heavy chain phosphorylation inhibited the actin-activated Mg-ATPase activity of Dictyostelium myosin II in 5-10 mM Mg2+ and low ionic strength through an increase in Kapp.     

Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial regulation of myosin II filament disassembly

Studies in Dictyostelium discoideum have established that the cycle of myosin II bipolar filament assembly and disassembly controls the temporal and spatial localization of myosin II during critical cellular processes, such as cytokinesis and cell locomotion. Myosin heavy chain kinase A (MHCK A) is a key enzyme regulating myosin II filament disassembly through myosin heavy chain phosphorylation in Dictyostelium. Under various cellular conditions, MHCK A is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. MHCK A possesses an aminoterminal coiled-coil domain that participates in the oligomerization, cellular localization, and actin binding activities of the kinase. In the current study, we show that the interaction between the coiled-coil domain of MHCK A and filamentous actin leads to an approximately 40-fold increase in the initial rate of kinase catalytic activity. Actin-mediated activation of MHCK A involves increased rates of kinase autophosphorylation and requires the presence of the coiled-coil domain. Structure-function analyses revealed that the coiled-coil domain alone binds to actin filaments (apparent K(D) = 0.9 microm) and thus mediates the direct interaction with F-actin required for MHCK A activation. Collectively, these results indicate that MHCK A recruitment to actin-rich sites could lead to localized activation of the kinase via direct interaction with actin filaments, and thus this mode of kinase regulation may represent an important mechanism by which the cell achieves localized disassembly of myosin II filaments required for specific changes in cell shape.     

Actin and myosin: control of filament assembly

Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35S-labelled actin monomers into unlabelled actin filaments, and exchange of [14C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca2+-dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.     

Biochemical characterization of a Dictyostelium myosin II heavy-chain phosphatase that promotes filament assembly.

In Dictyostelium cells, myosin II is found as cytosolic nonassembled monomers and cytoskeletal bipolar filaments. It is thought that the phosphorylation state of three threonine residues in the tail of myosin II heavy chain regulates the molecular motor's assembly state and localization. Phosphorylation of the myosin heavy chain at threonine residues 1823, 1833 and 2029 is responsible for maintaining myosin in the nonassembled state, and subsequent dephosphorylation of these residues is a prerequisite for assembly into the cytoskeleton. We report here the characterization of myosin heavy-chain phosphatase activities in Dictyostelium utilizing myosin II phosphorylated by myosin heavy-chain kinase A as a substrate. One of the myosin heavy-chain phosphatase activities was identified as protein phosphatase 2A and the purified holoenzyme was composed of a 37-kDa catalytic subunit, a 65-kDa A subunit and a 55-kDa B subunit. The protein phosphatase 2A holoenzyme displays two orders of magnitude higher activity towards myosin phosphorylated on the heavy chains than it does towards myosin phosphorylated on the regulatory light chains, consistent with a role in the control of filament assembly. The purified myosin heavy-chain phosphatase activity promotes bipolar filament assembly in vitro via dephosphorylation of the myosin heavy chain. This system should provide a valuable model for studying the regulation and localization of protein phosphatase 2A in the context of cytoskeletal reorganization.     

Determinants for substrate phosphorylation by Dictyostelium myosin II heavy chain kinases A and B and eukaryotic elongation factor-2 kinase

The alpha kinases are a widespread family of atypical protein kinases characterized by a novel type of catalytic domain. In this paper the peptide substrate recognition motifs for three alpha kinases, Dictyostelium discoideum myosin heavy chain kinase (MHCK) A and MHCK B and mammalian eukaryotic elongation factor-2 kinase (eEF-2K), were characterized by incorporating amino acid substitutions into a previously identified MHCK A peptide substrate (YAYDTRYRR) (Luo X. et al. (2001) J. Biol. Chem. 276, 17836-43). A lysine or arginine in the P+1 position on the C-terminal side of the phosphoacceptor threonine (P site) was found to be critical for peptide substrate recognition by MHCK A, MHCK B and eEF-2K. Phosphorylation by MHCK B was further enhanced 8-fold by a basic residue in the P+2 position whereas phosphorylation by MHCK A was enhanced 2- to 4-fold by basic residues in the P+2, P+3 and P+4 positions. eEF-2K required basic residues in both the P+1 and P+3 positions to recognize peptide substrates. eEF-2K, like MHCK A and MHCK B, exhibited a strong preference for threonine as the phosphoacceptor amino acid. In contrast, the Dictyostelium VwkA and mammalian TRPM7 alpha kinases phosphorylated both threonine and serine residues. The results, together with a phylogenetic analysis of the alpha kinase catalytic domain, support the view that the metazoan eEF-2Ks and the Dictyostelium MHCKs form a distinct subgroup of alpha kinases with conserved properties.     

Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.     

The carboxyl-terminal isoforms of smooth muscle myosin heavy chain determine thick filament assembly properties.

The alternatively spliced SM1 and SM2 smooth muscle myosin heavy chains differ at their respective carboxyl termini by 43 versus 9 unique amino acids. To determine whether these tailpieces affect filament assembly, SM1 and SM2 myosins, the rod region of these myosin isoforms, and a rod with no tailpiece (tailless), were expressed in Sf 9 cells. Paracrystals formed from SM1 and SM2 rod fragments showed different modes of molecular packing, indicating that the tailpieces can influence filament structure. The SM2 rod was less able to assemble into stable filaments than either SM1 or the tailless rods. Expressed full-length SM1 and SM2 myosins showed solubility differences comparable to the rods, establishing the validity of the latter as a model for filament assembly. Formation of homodimers of SM1 and SM2 rods was favored over the heterodimer in cells coinfected with both viruses, compared with mixtures of the two heavy chains renatured in vitro. These results demonstrate for the first time that the smooth muscle myosin tailpieces differentially affect filament assembly, and suggest that homogeneous thick filaments containing SM1 or SM2 myosin could serve distinct functions within smooth muscle cells.     

Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.     

Telokin/KRP differentially modulates myosin II filament assembly and regulatory light chain phosphorylation in fibroblasts

Transgenic 3T3 fibroblasts were made to express either wild-type telokin (KRP) or its truncated version lacking the C-terminal domain essential for binding to myosin. The content of myosin II filaments was markedly increased while regulatory light chain phosphorylation was decreased in the cells expressing KRP but not the C-truncated version. It could be concluded that (i) KRP promotes polymerization of nonmuscle myosin but reduces its RLC phosphorylation, (ii) these effects involve direct KRP binding to myosin, and (iii) KRP-expressing fibroblasts are a convenient model for assessing the role of myosin structural dynamics in cell motility.     

Dictyostelium myosin heavy chain kinase A regulates myosin localization during growth and development

Phosphorylation of the Dictyostelium myosin II heavy chain (MHC) has a key role in regulating myosin localization in vivo and drives filament disassembly in vitro. Previous molecular analysis of the Dictyostelium myosin II heavy chain kinase (MHCK A) gene has demonstrated that the catalytic domain of this enzyme is extremely novel, showing no significant similarity to the known classes of protein kinases (Futey, L. M., Q. G. Medley, G. P. Cote, and T. T. Egelhoff. 1995. J. Biol. Chem. 270:523-529). To address the physiological roles of this enzyme, we have analyzed the cellular consequences of MHCK A gene disruption (mhck A- cells) and MHCK A overexpression (MHCK A++ cells). The mhck A- cells are viable and competent for tested myosin-based contractile events, but display partial defects in myosin localization. Both growth phase and developed mhck A- cells show substantially reduced MHC kinase activity in crude lysates, as well as significant overassembly of myosin into the Triton-resistant cytoskeletal fractions. MHCK A++ cells display elevated levels of MHC kinase activity in crude extracts, and show reduced assembly of myosin into Triton-resistant cytoskeletal fractions. MHCK A++ cells show reduced growth rates in suspension, becoming large and multinucleated, and arrest at the mound stage during development. These results demonstrate that MHCK A functions in vivo as a protein kinase with physiological roles in regulating myosin II localization and assembly in Dictyostelium cells during both growth and developmental stages.     

Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation

Abnormalities in the huntingtin protein (Htt) are associated with Huntington's disease. Despite its importance, the function of Htt is largely unknown. We show that Htt is required for normal chemotaxis and cytokinesis in Dictyostelium discoideum. Cells lacking Htt showed slower migration toward the chemoattractant cAMP and contained lower levels of cortical myosin II, which is likely due to defects in dephosphorylation of myosin II mediated by protein phosphatase 2A (PP2A). htt(-) cells also failed to maintain myosin II in the cortex of the cleavage furrow, generating unseparated daughter cells connected through a thin cytoplasmic bridge. Furthermore, similar to Dictyostelium htt(-) cells, siRNA-mediated knockdown of human HTT also decreased the PP2A activity in HeLa cells. Our data indicate that Htt regulates the phosphorylation status of myosin II during chemotaxis and cytokinesis through PP2A.     

Multiple regulatory steps control mammalian nonmuscle myosin II assembly in live cells

To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (ΔIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Δtailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Δtailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.     

Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis

Myosin II is activated by the monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). Its ATPase activity is further enhanced by MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC). As these phosphorylated MRLCs are colocalized with their heavy chains at the contractile ring in dividing cells, we believe that the phosphorylated MRLC acts as a subunit of the activated myosin II during cytokinesis. However, the distinct role(s) of 1P- and 2P-MRLC during cytokinesis has not been elucidated. In this study, a monoclonal antibody (4F12) specific for 2P-MRLC was raised and used to examine the roles of 2P-MRLC in cultured mammalian cells. Our confocal microscopic observations using 4F12 revealed that 2P-MRLC localized to the contractile ring, and, unexpectedly, to the midzone also. Interestingly, 2P-MRLC did not colocalize with 1P-MRLC, myosin II heavy chain, and F-actin at the midzone. These results suggest that 2P-MRLC has a role different from that of 1P-MRLC at the midzone, and is not a subunit of myosin II.     

Developmental consequences of the lack of myosin heavy chain in Dictyostelium discoideum

Two different Dictyostelium discoideum cell lines that lack myosin heavy chain protein (MHC A) have been previously described. One cell line (mhcA) was created by antisense RNA inactivation of the endogenous mRNA and the other (HMM) by insertional mutagenesis of the endogenous myosin gene. The two cell lines show similar developmental defects; they are delayed in aggregation and become arrested at the mound stage. However, when cells that lack myosin heavy chain are mixed with wild-type cells, some of the mutant cells are capable of completing development to form mature spores. The pattern of expression of a number of developmentally regulated genes has been examined in both mutant cell lines. Although morphogenesis becomes aberrant before aggregation is completed, all of the markers that we have examined are expressed normally. These include genes expressed prior to aggregation as well as prespore genes expressed later in development. It appears that the signals necessary for cell-type differentiation are generated in the aborted structures formed by cells lacking MHC A. The mhcA cells have negligible amounts of MHC A protein while the HMM cells express normal amounts of a fragment of the myosin heavy chain protein similar to heavy meromyosin (HMM). The expression of myosin light chain was examined in these two cell lines. HMM cells accumulate normal amounts of the 18,000-D light chain, while the amount of light chain in mhcA cells is dramatically reduced. It is likely that the light chains assemble normally with the HMM fragment in HMM cells, while in cells lacking myosin heavy chain (mhcA) the light chains are unstable.     

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.     

Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum

A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.