MACF1
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Microtubule-actin cross-linking factor 1, isoforms 1/2/3/5 is a
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
that in humans is encoded by the ''MACF1''
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
. MACF1 encodes a large protein containing numerous
spectrin Spectrin is a cytoskeletal protein that lines the intracellular side of the plasma membrane in eukaryotic cells. Spectrin forms pentagonal or hexagonal arrangements, forming a scaffold and playing an important role in maintenance of plasma mem ...
and
leucine-rich repeat A leucine-rich repeat (LRR) is a protein structural motif that forms an α/β horseshoe tertiary structure, fold. It is composed of repeating 20–30 amino acid stretches that are unusually rich in the hydrophobic amino acid leucine. These Pr ...
(LRR) domains. MACF1 is a member of a family of proteins that form bridges between different
cytoskeletal The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all Cell (biology), cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane ...
elements. This protein facilitates
actin Actin is a family of globular multi-functional proteins that form microfilaments in the cytoskeleton, and the thin filaments in muscle fibrils. It is found in essentially all eukaryotic cells, where it may be present at a concentration of ...
-
microtubule Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27 nanometer, nm and have an inner diameter bet ...
interactions at the cell periphery and couples the microtubule network to
cellular junction Cell junctions or junctional complexes are a class of cellular structures consisting of multiprotein complexes that provide contact or adhesion between neighboring cells or between a cell and the extracellular matrix in animals. They also maintain ...
s. MACF1 belongs to a subset of +TIPs or proteins which bind to growing microtubule ends called spectraplakins. Spectraplakins characteristically have distinctive
microtubule Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27 nanometer, nm and have an inner diameter bet ...
and
actin Actin is a family of globular multi-functional proteins that form microfilaments in the cytoskeleton, and the thin filaments in muscle fibrils. It is found in essentially all eukaryotic cells, where it may be present at a concentration of ...
binding domains, which allow MACF1 to bind to both cytoskeletal elements. MACF1 goes by many names and is also called ACF7 or actin cross-linking factor 7, MACF, macrophin, trabeculin α, and ABP620. Alternatively spliced transcript variants encoding distinct isoforms of MACF1 have been described. MACF1 is also an important protein for
cell migration Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryogenesis, embryonic development, wound healing and immune system, immune responses all require the orchestrated movemen ...
in processes such as wound healing.


Structure

MACF1 is an enormous protein of 5380 amino acid residues. The
N-terminal The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...
segment has an actin binding domain and the
C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...
segment has a +TIP binding site as well as microtubule interacting domains. This allows MACF1 to crosslink both actin and microtubules. The C-terminal region contains both a Gas2-related domain and a GSR-repeat domain, which both are involved with interacting with microtubules. The C-terminus of MACF1 is thought to associate to the microtubule lattice through the acidic C-terminal tails of
tubulin Tubulin in molecular biology can refer either to the tubulin protein superfamily of globular proteins, or one of the member proteins of that superfamily. α- and β-tubulins polymerize into microtubules, a major component of the eukaryotic cytosk ...
subunits. However, MACF1 does not always associate with the microtubule directly, and also binds through many proteins which localize at the microtubule plus end. Such proteins include EB1,
CLASP1 Cytoplasmic linker associated protein 1, also known as CLASP1, is a protein which in humans is encoded by the ''CLASP1'' gene. Function CLASP1 belongs to a family of microtubule-associated proteins involved in attachment of microtubules to th ...
, and
CLASP2 Cytoplasmic linker associated protein 2, also known as CLASP2, is a protein which in humans is encoded by the ''CLASP2'' gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is ...
, whose interactions with MACF1 were determined through coimmunoprecipitation assay. Not only does MACF1's C-terminal tail bind to microtubules, but it also has key
phosphorylation In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...
sites. When these sites are phosphorylated by its regulator
GSK3β Glycogen synthase kinase-3 beta, (GSK-3 beta), is an enzyme that in humans is encoded by the ''GSK3B'' gene. In mice, the enzyme is encoded by the Gsk3b gene. Abnormal regulation and expression of GSK-3 beta is associated with an increased susce ...
, the ability of MACF1 to bind to microtubules is disrupted. MACF1 also has an actin-regulated
ATPase ATPases (, Adenosine 5'-TriPhosphatase, adenylpyrophosphatase, ATP monophosphatase, triphosphatase, ATP hydrolase, adenosine triphosphatase) are a class of enzymes that catalyze the decomposition of ATP into ADP and a free phosphate ion or ...
domain, which is approximately 3000 amino acid residues long in the C-terminal region, and is responsible for cytoskeletal dynamics.


Function


Embryonic development

MACF1 is important for
embryonic development In developmental biology, animal embryonic development, also known as animal embryogenesis, is the developmental stage of an animal embryo. Embryonic development starts with the fertilization of an egg cell (ovum) by a sperm, sperm cell (spermat ...
. For mice, by embryonic day 7.5 (E7.5), MACF1 is expressed in the headfold and
primitive streak The primitive streak is a structure that forms in the early embryo in amniotes. In amphibians, the equivalent structure is the blastopore. During early embryonic development, the embryonic disc becomes oval shaped, and then pear-shaped with the ...
, and by E8.5 the protein is expressed in neuronal tissues and the
foregut The foregut in humans is the anterior part of the alimentary canal, from the distal esophagus to the first half of the duodenum, at the entrance of the bile duct. Beyond the stomach, the foregut is attached to the abdominal walls by mesentery. ...
. MACF1 was shown to be present in the
Wnt signaling In cellular biology, the Wnt signaling pathways are a group of signal transduction pathways which begin with proteins that pass signals into a cell through cell surface receptors. The name Wnt, pronounced "wint", is a portmanteau created from the ...
pathway. When Wnt signalling is not present, MACF1 associates with a complex containing axin,
β-catenin Catenin beta-1, also known as β-catenin (''beta''-catenin), is a protein that in humans is encoded by the ''CTNNB1'' gene. β-Catenin is a dual function protein, involved in regulation and coordination of cell–cell adhesion and gene transcr ...
,
GSK3β Glycogen synthase kinase-3 beta, (GSK-3 beta), is an enzyme that in humans is encoded by the ''GSK3B'' gene. In mice, the enzyme is encoded by the Gsk3b gene. Abnormal regulation and expression of GSK-3 beta is associated with an increased susce ...
, and APC. However, upon Wnt signaling, MACF1 is involved in a translation and binding of the axin complex to LTP6 at the
cell membrane The cell membrane (also known as the plasma membrane or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of a cell from the outside environment (the extr ...
. Also, MACF1 is required for sufficient β-catenin to travel to the nucleus, where subsequently TCF/β-catenin-dependent transcriptional activation of a gene T encoding the protein
brachyury T-box transcription factor T, also known as Brachyury protein, is encoded for in humans and other apes by the ''TBXT'' gene. Brachyury functions as a transcription factor within the T-box, T-box family of genes. Brachyury Sequence homology, homo ...
occurs. Brachyury is an essential transcription factor required for mesoderm formation. Without MACF1, insufficient brachyury is transcribed, and hence, the
mesoderm The mesoderm is the middle layer of the three germ layers that develops during gastrulation in the very early development of the embryo of most animals. The outer layer is the ectoderm, and the inner layer is the endoderm.Langman's Medical ...
does not form. In fact, MACF1 knock-out mice, which lack the protein, show clear developmental retardation by E7.5, and eventually die at
gastrulation Gastrulation is the stage in the early embryonic development of most animals, during which the blastula (a single-layered hollow sphere of cells), or in mammals, the blastocyst, is reorganized into a two-layered or three-layered embryo known as ...
due to defects in the formation of the primitive streak, node, and mesoderm.


Cell migration

Mice with conditional knock-outs in MACF1 in hair follicle stem cells have defects in cell migration. The
focal adhesions In cell biology, focal adhesions (also cell–matrix adhesions or FAs) are large macromolecular assemblies through which mechanical force and regulatory signals are transmitted between the extracellular matrix (ECM) and an interacting cell. Mor ...
in cells lacking MACF1 associate with cables of F-actin, causing
cell migration Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryogenesis, embryonic development, wound healing and immune system, immune responses all require the orchestrated movemen ...
to stall. Wild-type cells with MACF1 present have coordinated cytoskeletal dynamics, which allow for proper cell migration. MACF1 plays an important role in microtubule organization, and without MACF1, microtubules in migrating cells are bending and curly, instead of straight and radial. When wounded, conditional knock-outs for MACF1 have around a 40% delay in migration over 4 to 6 days after injury compared to the wild-type controls, showing that MACF1 plays an important role in cell migration. There are suggestions that imply that MACF1 may play a role in golgi polarization. The major known regulator of MACF1 is
GSK3β Glycogen synthase kinase-3 beta, (GSK-3 beta), is an enzyme that in humans is encoded by the ''GSK3B'' gene. In mice, the enzyme is encoded by the Gsk3b gene. Abnormal regulation and expression of GSK-3 beta is associated with an increased susce ...
, which when uninhibited phosphorylates MACF1 among its many other substrates and uncouples MACF1 from microtubules. The phosphorylation of MACF1 occurs in the GSR domain, which is involved in microtubule binding, and has 32% of the amino acid residues are serines or threonines. MACF1 has 6 serines, which are possible GSK3β phosphorylation sites. GSK3β activity is high in non-stimulated cells, but during
cell migration Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryogenesis, embryonic development, wound healing and immune system, immune responses all require the orchestrated movemen ...
its activity is dampened along the cell leading edge. In vivo, GSK3β activity is inhibited by Wnt signalling, but in vitro it is typically inhibited by
cdc42 Cell division control protein 42 homolog (Cdc42 or CDC42) is a protein that in humans is encoded by the ''CDC42'' gene. Cdc42 is involved in regulation of the cell cycle. It was originally identified in ''S. cerevisiae'' (yeast) as a mediator of ...
. Extracellular Wnt signalling acts on the
Frizzled Frizzled is a family of atypical G protein-coupled receptor, G protein-coupled receptors that serve as receptors in the Wnt signaling pathway and other signaling pathways. When activated, Frizzled leads to activation of Dishevelled in the cytosol ...
receptor on the cellular membrane, which then, through a signalling cascade inhibits GSK3β. The inhibition of GSK3β creates a gradient at the leading edge, allowing MACF1 to remain active and unphosphorylated, so that it can form necessary connections between microtubules and actin so migration can occur. It was found in hair follicle stem cells that phosphorylation-refractile MACF1 rescues microtubule architecture from a MACF1 knock-out, whereas phosphorylation-constitutive MACF1 is unable to rescue the phenotype. However, neither phosphorylation-refractile MACF1 nor phosphorylation-constitutive MACF1 are able to rescue polarized cell movement. This implies that the phospho-regulation dynamics permitted in the wild type MACF1 are necessary for polarized cell movement to take place.


Clinical significance

In breast carcinoma cells, addition of heregulin β activates
ErbB2 Receptor tyrosine-protein kinase erbB-2 is a protein that normally resides in the membranes of cells and is encoded by the ''ERBB2'' gene. ERBB is abbreviated from erythroblastic oncogene B, a gene originally isolated from the avian genome. The ...
, a receptor tyrosine, which causes microtubules to form many cell protrusions to cause cell motility. ErbB2 controls microtubule outgrowth and stabilization at the cell cortex through a specific pathway. When
GSK3β Glycogen synthase kinase-3 beta, (GSK-3 beta), is an enzyme that in humans is encoded by the ''GSK3B'' gene. In mice, the enzyme is encoded by the Gsk3b gene. Abnormal regulation and expression of GSK-3 beta is associated with an increased susce ...
is active, APC and
CLASP2 Cytoplasmic linker associated protein 2, also known as CLASP2, is a protein which in humans is encoded by the ''CLASP2'' gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is ...
are sequentially inactivated by the
kinase In biochemistry, a kinase () is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the high-energy ATP molecule don ...
, which gives a condition where microtubule formation is not favoured at the front of the cell. For cell migration to occur, a mechanism is needed to decrease the activity of GSK3β to promote growth of microtubules. First, ErbB2 recruits Memo (mediator of ErbB2-driven motility) to the plasma membrane, which then promotes the phosphorylation of GSK3β on serine 9. This decreases the amount of GSK3β activity, and permits the localization of APC and CLASP2 to the cell membrane, which are both microtubule +TIPs. Although CLASP2 is present at the cell membrane, it appears to have a separate, independent mechanism for microtubule growth than APC. When ErbB2 inactivates GSK3β, APC localizes to the membrane and is then able to recruit MACF1 to the membrane as well. The APC-mediated recruitment of MACF1 to the membrane is required and sufficient for microtubule capture and stabilization at the cell cortex during breast carcinoma cell motility.


References


Further reading

* * * * * * * * * * * * * * * {{refend EF-hand-containing proteins Plakins