Hanna Brzeska

Regulation of Class I and Class II Myosins by Heavy Chain Phosphorylation

Myosins have been traditionally viewed as mechanochemical, actin-activated MgATPases that convert the energy of ATP hydrolysis into force between actin and myosin filaments exhibited as either movement (isotonic contraction) or tension (isometric contraction). “Conventional” myosins, i.e.myosins that form filaments, consist of a pair of heavy chains (;200 kDa) and two pairs of light chains, the regulatory light chain and the essential light chain. Each heavy chain has an N-terminal, globular head, where the actin-activated ATPase activity resides, and a C-terminal tail, through which the two heavy chains interact to form an a-helical coiled-coil rod. One of each pair of light chains is bound to the neck region of the globular heads (subfragment-1) near the head-tail junction. The tail also mediates the self-association of multiple myosin molecules into bipolar thick filaments. Ca activates muscle actomyosins predominantly in one of two general ways (1, 2): (i) actin-based regulation in which Ca binds to troponin C, a component of the tropomyosin-troponin complex that lies in the groove of the a-helical coiled-coil actin thin filament (skeletal muscle); (ii) myosin light chain-based regulation in which either Ca/calmodulin-dependent myosin light chain kinase phosphorylates a serine in the regulatory light chain (smooth muscle) or Ca binds directly to the regulatory light chain (molluscan muscle). For many years, myosin filaments, as well as actin filaments, were thought to be essential for physiologically meaningful interactions of actin and myosin. The discovery of monomeric Acanthamoeba myosin I (3), however, proved that single-headed, nonfilamentous myosins, i.e. “unconventional” myosins, could exert force between, and translocate along, actin filaments. This expanded the potential physiological roles for myosins beyond those that might be performed by filamentous myosins and initiated a search for other unconventional myosins. We now recognize the existence of a myosin family presently comprising 11 classes defined by the extent of sequence homology within the subfragment-1 (S-1) domain: 10 unconventional myosin classes and 1 conventional (so named because it was the first to be discovered) class. There are several excellent recent reviews of the sequence, structural diversity, and possible biological functions of the multiple members of this extended family (1, 4–6). Most conventional (class II, by the current classification system) nonmuscle myosins are regulated by phosphorylation of their regulatory light chains, similarly to smooth muscle myosin (7). All unconventional myosins contain at least one light chain which, with the notable exception of the amoeba myosins, appears to be calmodulin (4, 8). In some cases, calmodulin has been shown to be associated with the purified myosin, but in many cases a calmodulin light chain is inferred from the heavy chain sequence, which can contain from one to six calmodulin-binding IQ motifs in the light chain-binding region (4, 8). The IQ motifs and the regulation of unconventional myosins by Ca interacting with calmodulin, which is reminiscent even if different in detail (e.g. in some cases Ca causes calmodulin to dissociate from the heavy chain with loss of actomyosin activities) of the regulation of molluscan myosins, were reviewed recently (4, 8, 9) and will not be discussed here. Early in the study of the Acanthamoeba myosins it became clear that they were regulated exclusively by a heretofore unknown mechanism: heavy chain phosphorylation (10). The phosphorylation occurs in the S-1 domain of the unconventional, class I myosins, as one might expect for a covalent modification that regulates MgATPase activity, but, and quite unexpectedly, near or at the end of the C-terminal tail of the conventional, class II myosin (6). The latter modification is quite remote from the regulated ATPase site in the S-1 domain and separated from it by a relatively rigid a-helical coiled-coil, thus posing some very interesting questions about the mechanism of signal transduction between the phosphorylation site and the catalytic site. Thus far, only Acanthamoeba, Dictyostelium, and Physarummyosins have been shown definitively to be regulated by heavy chain phosphorylation. However, there are numerous examples of heavy chain phosphorylation of vertebrate nonmuscle class II myosins, in vivo as well as in vitro, with as yet no known substantial biochemical or physiological consequences (6, 11). Heavy chain phosphorylation of vertebrate nonmuscle class II myosins deserves more attention than it has received. Possibly this review will stimulate interest in those systems. In addition to novel mechanisms for regulation of actomyosin MgATPase activity, an emerging feature from the study of unconventional myosins is the probable role of their nonfilamentous tails in regulating function. Tail domains may determine the intracellular localization of the myosin and the physiological task for which the mechanochemical activity of its S-1 domain is used. This aspect of unconventional myosins will be briefly addressed.

Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essential in vivo function(s)

The single class I myosin (MYOA) of Aspergillus nidulans is essential for hyphal growth. It is generally assumed that the functions of all myosins depend on their actin-activated MgATPase activity. Here we show that MYOA mutants with no more than 1% of the actin-activated MgATPase activity of wild-type MYOA in vitro and no detectable in vitro motility activity can support fungal cell growth, albeit with a delay in germination time and a reduction in hyphal elongation. From these and other data, we conclude that the essential role(s) of myosin I in A. nidulans is probably structural, requiring little, if any, actin-activated MgATPase or motor activity, which have long been considered the defining characteristics of the myosin family.

An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins: APPLICATION TO CLASS I MYOSINS, PAKS, AND CARMIL

Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro.

Acanthamoeba myosin IC colocalizes with phosphatidylinositol 4,5-bisphosphate at the plasma membrane due to the high concentration of negative charge.

The tail of Acanthamoeba myosin IC (AMIC) has a basic region (BR), which contains a putative pleckstrin homology (PH) domain, followed by two Gly/Pro/Ala (GPA)-rich regions separated by a Src homology 3 (SH3) domain. Cryoelectron microscopy had shown that the tail is folded back on itself at the junction of BR and GPA1, and nuclear magnetic resonance spectroscopy indicated that the SH3 domain may interact with the putative PH domain. The BR binds to acidic phospholipids, and the GPA region binds to F-actin. We now show that the folded tail does not affect the affinity of AMIC for acidic phospholipids. AMIC binds phosphatidylinositol 4,5-bisphosphate (PIP2) with high affinity (∼1 μm), but binding is not stereospecific. When normalized to net negative charge, AMIC binds with equal affinity to phosphatidylserine (PS) and PIP2. This and other data show that the putative PH domain of AMIC is not a typical PIP2-specific PH domain. We have identified a 13-residue sequence of basic-hydrophobic-basic amino acids within the putative PH domain that may be a major determinant of binding of AMIC to acidic phospholipids. Despite the lack of stereospecificity, AMIC binds 10 times more strongly to vesicles containing 5% PIP2 plus 25% PS than to vesicles containing only 25% PS, suggesting that AMIC may be targeted to PIP2-enriched regions of the plasma membrane. In agreement with this, AMIC colocalizes with PIP2 at dynamic, protrusive regions of the plasma membrane. We discuss the possibility that AMIC binding to PIP2 may initiate the formation of a multiprotein complex at the plasma membrane.

[2] Purification of myosin I and myosin I heavy chain kinase from Acanthamoeba castellanii

Publisher Summary The myosins I are structurally distinct from conventional myosins (myosins II) in that they are globular, single-headed proteins whose native molecular masses range from 140,000 to 160,000 Da. The activity of the myosin I heavy chain kinase is enhanced by its autophosphorylation which in turn is stimulated by phosphatidylserine. Myosin I and its heavy chain kinase are isolated from Acanthamoeba castellanii by conventional chromatographic methods. This chapter presents detailed procedures for isolating both. A cell extract is adsorbed to DE-52 and the kinase and myosin I are step eluted in a single pool. This material is applied to a phosphocellulose column, which is then eluted with a salt gradient in order to resolve the kinase and all three myosin I isozymes. The myosins I are further purified (individually) on ADP- or ATP-agarose and Mono Q columns, both eluted with salt gradients. Myosin I is assayed during its purification by its (K + ,EDTA)-ATPase activity. A radioisotopic form of this assay utilizing [γ- 32 P]ATP is necessary, at least in the early stages, because of the presence of phosphate liberated from pyrophosphate. It is difficult to estimate recovery and the extent of purification, but this procedure should yield 500 μg or more of myosin I heavy chain kinase.

Functional analysis of tail domains of Acanthamoeba myosin IC by characterization of truncation and deletion mutants.

Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single 17-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of myosin IC exhibits triphasic actin dependence, apparently as a consequence of the two actin-binding sites, and is regulated by phosphorylation of Ser-329 in the head. The 50-kDa tail consists of a basic domain, a glycine/proline/alanine-rich (GPA) domain, and aSrc homology 3 (SH3) domain, often referred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the functions of the tail domains more precisely, we determined the properties of expressed wild type and six mutant myosins, an SH3 deletion mutant and five mutants truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head domains, respectively. We found that both the TH-1 and GPA-2 domains bind F-actin in the presence of ATP. Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of the SH3 domain had no effect. Also, none of the tail domains, including the SH3 domain, affected either the K m orV max for the phosphorylation of Ser-329 by myosin I heavy chain kinase.

Molecular Basis of Dynamic Relocalization of Dictyostelium Myosin IB

Background: Class I myosins contribute to membrane-associated events. Results: A short segment of basic/hydrophobic amino acids in the tail which binds acidic phospholipids and the actin binding site in the head is required for relocalization of Dictyostelium myosin IB. Conclusion: Dynamic relocalization results from competition between membrane acidic phospholipids and cytoplasmic F-actin. Significance: The molecular basis of myosin I relocation is fundamental to understanding cell motility. Class I myosins have a single heavy chain comprising an N-terminal motor domain with actin-activated ATPase activity and a C-terminal globular tail with a basic region that binds to acidic phospholipids. These myosins contribute to the formation of actin-rich protrusions such as pseudopodia, but regulation of the dynamic localization to these structures is not understood. Previously, we found that Acanthamoeba myosin IC binds to acidic phospholipids in vitro through a short sequence of basic and hydrophobic amino acids, BH site, based on the charge density of the phospholipids. The tail of Dictyostelium myosin IB (DMIB) also contains a BH site. We now report that the BH site is essential for DMIB binding to the plasma membrane and describe the molecular basis of the dynamic relocalization of DMIB in live cells. Endogenous DMIB is localized uniformly on the plasma membrane of resting cells, at active protrusions and cell-cell contacts of randomly moving cells, and at the front of motile polarized cells. The BH site is required for association of DMIB with the plasma membrane at all stages where it colocalizes with phosphoinositide bisphosphate/phosphoinositide trisphosphate (PIP2/PIP3). The charge-based specificity of the BH site allows for in vivo specificity of DMIB for PIP2/PIP3 similar to the PH domain-based specificity of other class I myosins. However, DMIB-head is required for relocalization of DMIB to the front of migrating cells. Motor activity is not essential, but the actin binding site in the head is important. Thus, dynamic relocalization of DMIB is determined principally by the local PIP2/PIP3 concentration in the plasma membrane and cytoplasmic F-actin.

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.

Selective localization of myosin-I proteins in macropinosomes and actin waves

Class I myosins are widely expressed with roles in endocytosis and cell migration in a variety of cell types. Dictyostelium express multiple myosin Is, including three short‐tailed (Myo1A, Myo1E, Myo1F) and three long‐tailed (Myo1B, Myo1C, Myo1D). Here we report the molecular basis of the specific localizations of short‐tailed Myo1A, Myo1E, and Myo1F compared to our previously determined localization of long‐tailed Myo1B. Myo1A and Myo1B have common and unique localizations consistent with the various features of their tail region; specifically the BH sites in their tails are required for their association with the plasma membrane and heads are sufficient for relocalization to the front of polarized cells. Myo1A does not localize to actin waves and macropinocytic protrusions, in agreement with the absence of a tail region which is required for these localizations of Myo1B. However, in spite of the overall similarity of their domain structures, the cellular distributions of Myo1E and Myo1F are quite different from Myo1A. Myo1E and Myo1F, but not Myo1A, are associated with macropinocytic cups and actin waves. The localizations of Myo1E and Myo1F in macropinocytic structures and actin waves differ from the localization of Myo1B. Myo1B colocalizes with F‐actin in the actin waves and at the tips of mature macropinocytic cups whereas Myo1E and Myo1F are in the interior of actin waves and along the entire surface of macropinocytic cups. Our results point to different mechanisms of targeting of short‐ and long‐tailed myosin Is, and are consistent with these myosins having both shared and divergent cellular functions.

Mammalian Nonmuscle Myosin II Binds to Anionic Phospholipids with Concomitant Dissociation of the Regulatory Light Chain

Mammalian cells express three Class II nonmuscle myosins (NM): NM2A, NM2B, and NM2C. The three NM2s have well established essential roles in cell motility, adhesion, and cytokinesis and less well defined roles in vesicle transport and other processes that would require association of NM2s with cell membranes. Previous evidence for the mechanism of NM2-membrane association includes direct interaction of NM2s with membrane lipids and indirect interaction by association of NM2s with membrane-bound F-actin or peripheral membrane proteins. Direct binding of NM2s to phosphatidylserine-liposomes, but not to phosphatidylcholine-liposomes, has been reported, but the molecular basis of the interaction between NM2s and acidic phospholipids has not been previously investigated. We now show that filamentous, full-length NM2A, NM2B, and NM2C and monomeric, non-filamentous heavy meromyosin bind to liposomes containing one or more acidic phospholipids (phosphatidylserine, phosphatidylinositol 4,5-diphosphate, and phosphatidylinositol 3,4,5-triphosphate) but do not bind to 100% phosphatidylcholine-liposomes. Binding of NM2s to acidic liposomes occurs predominantly through interaction of the liposomes with the regulatory light chain (RLC) binding site in the myosin heavy chain with concomitant dissociation of the RLC. Phosphorylation of myosin-bound RLC by myosin light chain kinase substantially inhibits binding to liposomes of both filamentous NM2 and non-filamentous heavy meromyosin; the addition of excess unbound RLC, but not excess unbound essential light chain, competes with liposome binding. Consistent with the in vitro data, we show that endogenous and expressed NM2A associates with the plasma membrane of HeLa cells and fibrosarcoma cells independently of F-actin.