Martina Costanzo
Maria Chiara Crosetto
A family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Kindlin-1 is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. Different studies show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms. Therefore, the KS is the first human skin fragility disorder caused by deficiency of a β1 integrin-associated focal adhesion protein. However, very little was known about native kindlin-1 or its physiological functions or the phenotypic consequences of its alterations in KS. (Kindlin-1 Is a Phosphoprotein Involved in Regulation of Polarity, Proliferation, and Motility of Epidermal Keratinocytes)
Wikipedia
KINDLIN PROTEINS
DEFINITION
Kindlins are a group of proteins implicated in integrins activation and functions into epidermal cells.
Kindlins consist of three evolutionarily conserved members, namely kindlin-1, kindlin-2 and kindlin-3. These proteins share considerable sequence and structural similarities.
THE GENE
They are encoded by three different genes, namely KIND1 (chromosome 20p12.3), KIND2 (chromosome 14q22.1) and KIND3 (chromosome 11q13.1). Kindlin-2 has ~62% homology with kindlin-1; kindlin-3 shares ~49% similarity with kindlin-1. They each have a pleckstrin homology (PH) domain flanked by a bipartite FERM domain located in the C-terminal region, unlike talin which has an N-terminal FERM domain. In addition, the F3 subdomain of the FERM domains of all three kindlins crucially contains a phosphotyrosine binding (PTB) fold resembling that of talin. The N-terminal region mediates interactions with ILK and migfilin while the F3 subdomain interacts with β1 and β3 integrins.
(Functions of Kindlin-1 in Human Keratinocytes and its Immunolocalization in Human Oral Mucosal Tissues)
CHEMICAL STRUCTURE AND IMAGES
Kindlins directly interact with β1, β2, and β3 integrins. All three kindlins interact with β1 and β3 integrin tails, while kindlin-3 has also been shown to bind β2 tails. F3 subdomain of kindlin bind a membrane proximal NPxY and a membrane distal NxxY motif on β integrin cytoplasmic tails. Data from Moser et al suggests that kindlin prefers to interact with a membrane distal (T/S)TxxNxxY amino acid sequence. Kindlins bind the distal NxxY .in β integrin tails and require the double Thr (in β1 tails) or Ser–Thr (in β3 tails) that are located between the two NxxY motifs for binding.
Figure. A model of kindlin binding to b integrin cytoplasmic tails.
Comparison of kindlin PH domain structures. The kindlin-1 PH domain structure reported here (top; with aromatic residues projecting into the binding pocket shown in green on the right), the apo (left) (72) and Ins(1,3,4,5)P4-bound NMR structure (equivalent to Ins(3,4,5)P3 binding physiologically) (right) (24) of the kindlin-2 PH domain (middle), and the kindlin-3 PH domain structure (bottom) (PDB ID 2YS3). The residues involved in the native salt bridge are shown for the kindlin-1 structure, and the structural equivalents for the other PH domain; the alternative salt bridge partner Glu450 is also shown for kindlin-1 and in the InsP-bound structure of the kindlin-2 domain, the interacting side chains other than His419 and Lys383 are shown in white.
Structure from PDB
* Kindlin-1
* kindlin-2
* Kindlin-3
CELLULAR FUNCTIONS AND REGULATION
Kindlins are implicated in integrins activation and functions into epidermal cells.
Integrins are key components of focal adhesions and represent dynamic protein complexes involved in cell migration and adhesion; they connect the cell to the external environment and mediate bidirectional signaling across the cell membrane. Integrins are glycosylated transmembrane heterodimeric adhesion receptors that are formed by non-covalently bound α and β subunits. Each integrin subunit consists of a large extracellular domain of about 700 amino acid residues (80–150 kDa), a single trans membrane α helix (~20 residues) and a short cytoplasmic domain of about 10–70 residues. In eukaryotes, there are 18 α and 8 β subunits which non-covalently heterodimerize to form 24 distinct integrins.
(The Kindlin protein family: new members to the club of focal adhesion proteins, 2009)
The extracellular domain of integrins interacts with extracellular matrix (ECM) proteins such as fibronectin and laminins while the short cytoplasmic tails mediate connections with the actin cytoskeleton via adaptor proteins such as talin. Inactive integrins are in a low affinity binding state and assume a bent conformation. Active integrin is characterized by an extended conformation although in some circumstances the bent conformation is also able to bind ligand with high affinity. Binding of several adaptor proteins to the cytoplasmic domain of integrins is a critical step in integrin activation.
Talin, a major cytoskeletal protein consisting of a N-terminal globular head and a C-terminal rod domain, is the first identified and best studied adaptor protein involved in integrin activation in several classes of integrins. The head domain of talin consists of a FERM domain (four point one protein, ezrin, radixin and moesin), a clover-shaped structure subdivided into F1, F2 and F3 subdomains. The F2 and F3 subdomains can both bind to β integrin although the F3 subdomain binds with higher affinity and has been shown to be the activating fragment of talin. The binding of talin FERM domain with the cytoplasmic tail of β integrin allows separation of both the α and βsubunits, resulting in increased affinity for extracellular ligand binding and integrin activation.
Kindlin family members can also bind to migfilin and ILK, two focal adhesion (FA) proteins that directly or indirectly regulate actin dynamics and intracellular signaling pathways.
Migfilin is composed of three C-terminal LIM domains, which mediate the interaction with kindlin-2 and are essential for its recruitment to cell– matrix adhesions; a central proline-rich region that binds to vasodilator-stimulated phosphoprotein (VASP) and an N-terminal region that binds to filamin.
ILK is composed of three structurally distinct protein domains: ankyrin-repeats at the N-terminus and a putative kinase domain situated C-terminally separated by a PH-like domain, which mediates binding to PtdIns(3,4,5)P3. ILK binds to Pinch-1 and Pinch-2 (particularly interesting new cysteine–histidine protein) via its first ankyrin-repeat and to the actin-binding proteins parvin and paxillin with its kinase domain. Furthermore, ILK has been shown to interact with the cytoplasmic tails of β1 and β3 integrins. However, the exact binding site is not mapped, nor is it clear whether ILK binds to β tails directly or indirectly. Its role as a serine/threonine kinase in vivo is also very controversial.
(The Kindlin protein family: new members to the club of focal adhesion proteins, 2009)
Kindlins and ILK are present in different subcellular compartments including cell–cell adhesions, the nucleus, and cell–matrix adhesions. Talin and kindlins bind β integrin cytoplasmic domains (1), which shift integrins from a low affinity state to a high affinity conformation for their ligands (2). Once integrins are in their active conformation, additional protein–protein interactions are established that link integrins with the cytoskeleton and growth factor signaling pathways. ILK and kindlins orchestrate these processes by interacting with each other and by recruiting other proteins to focal adhesions (3). ILK binds Pinch and the F-actin-binding proteins parvin and paxillin. In addition to its interaction with ILK, kindlins recruit the adaptor protein migfilin to focal adhesions, which in turn binds filamin. a: a subunit of integrins; b: b subunit of integrins; ECM: extracellular matrix; ILK: integrinlinked kinase; Pinch: particularly interesting new Cys–His protein. (The Kindlin protein family: new members to the club of focal adhesion proteins, 2009)
Kindlin-1 regulates cell shape and migration by controlling lamellipodia formation.
The lamellipodium is a cytoskeletal protein actin projection on the mobile edge of the cell. It contains a quasi-two-dimensional actin mesh; the whole structure propels the cell across a substrate. Within the lamellipodia are ribs of actin called microspikes, which, when they spread beyond the lamellipodium frontier, are called filopodia. The lamellipodium is born of actin nucleation in the plasma membrane of the cell and is the primary area of actin incorporation or microfilament formation of the cell. (Wikipedia)
Kindlin-1 forms molecular complexes with β1 integrin, α actinin, migfilin, and focal adhesion kinase and governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin.
(Kindlin-1 Is Required for RhoGTPase-Mediated Lamellipodia Formation in Keratinocytes)
Besides their role in cell–matrix adhesions, kindlins might also function in other cellular compartments. Kindlins are not exclusively found at integrin adhesion sites. In skin, kindlin-1 participates in forming stable junctions between the epidermis and the underlying basement membrane within the dermatoepidermal junction (DEJ) zone. In addition, kindlins have also been detected in the nucleus of epithelial and mesenchymal cells. Migfilin, a known binding partner of kindlins, has been demonstrated to function as a transcriptional cofactor and might aid in translocating kindlins to the nucleus. (The Kindlin protein family: new members to the club of focal adhesion proteins, 2009)
Figure. Possible roles of kindlin proteins in various cell compartments. Kindlins might function in various cellular compartments. Several kindlin binding partners have been identified. These include b integrin cytoplasmic tails, ILK and migfilin. All of these proteins localize to cell–matrix adhesions. Within these structures, kindlins are thought to mediate integrin activation. They might also dock the b integrin cytoplasmic tail to the actin cytoskeleton via a migfilin/filamin intermediary complex. In addition, kindlins have been shown to interact with ILK to promote adhesion strengthening. Besides their role in cell–matrix adhesions, kindlins might also function in other cellular compartments. Although the data are preliminary, immunostaining suggest that kindlins might localize to adherens junctions. In addition, kindlins have also been detected in the nucleus of epithelial and mesenchymal cells. Migfilin, a known binding partner of kindlins, has been demonstrated to function as a transcriptional cofactor and might aid in translocating kindlins to the nucleus. (The Kindlin protein family: new members to the club of focal adhesion proteins, 2009)
DIAGNOSTIC USE
CLINICAL FEATURES OF KINDLER SYNDROME
The main clinical features of KS are trauma-induced skin blistering predominantly involving acral sites and progressive poikiloderma and mucosal inflammation. Some individuals with KS suffer with varying degrees of photosensitivity,which often lessens with age. The skin blistering also has a tendency to subside with age although this is not a universal finding. Marked skin atrophy develops early in life (under the age of 5) especially on the dorsal aspects of the hands and feet and, in most cases, becomes generalized by adolescence. Gingivitis and periodontitis are also prominent features. Mucosal stenoses involving the esophagus, vagina, anus, and urethra have been reported. Gastrointestinal symptoms,including constipation and severe colitis, can also occur in KS. Other features may include pseudosyndactyly, conjunctivitis, mandibular abnormalities and orogenital leukokeratosis, although the diagnosis of KS in many reports detailing these clinical findings has not been substantiated by molecular analysis. There is also an increased of nonmelanoma skin cancer in KS with squamous cell carcinomas reported in acral skin or the mouth.
(Functions of Kindlin-1 in Human Keratinocytes and its Immunolocalization in Human Oral Mucosal Tissues)
ABNORMALITIES OF KERATINOCYTES:
In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis.
Kindlin-1-deficient epidermis exhibited almost no reactivity with the proliferation marker Ki67, in contrast to age-matched control skin, indicating that absence of kindlin-1 leads to decreased keratinocyte proliferation. The intensity of keratin 5, a marker of the intermediate filament system in proliferating epidermal keratinocytes, was substantially lower in kindlin-1 deficient skin, correlating with the Ki67 signal. In particular, only the basal layer of keratinocytes was stained with antibodies against keratin 5, whereas in normal control skin two or three suprabasal cell layers were positive. In contrast, epidermal differentiation was similar in KS and control skin, as assessed by keratin 10 and involucrin staining. Furthermore, kindlin-1 deficiency perturbed positioning of transmembrane components in basal keratinocytes. Instead of a preferential localization at the basal plasma membrane in control cells, collagen XVII was equally distributed all around the basal keratinocytes, also at the apical plasma membrane and in the cytoplasm, implicating loss of polarization). Integrin α6, which are targeted to the ventral surface of basal keratinocytes in control skin, were strongly reduced along some DEJZ stretches, while in others the deposition seemed increased. In addition, integrin α6 was also found at the lateral and apical cell membranes in KS skin, co-localizing with α3 integrin. So, histological feauters are epidermal atrophy, focal vacuolization of basal layer, pigmentary incontinence in upper dermis, mild lymphocytic infiltrate, extensive reduplication and disruption of lamina densa along the dermoepidermal junction beneath basal cells, cleft formation in lamina lucida. Desmosomes, hemidesmosomes, tonofilaments, anchoring filaments and fibrils appear normal and abundant keratin bodies present focally in the dermis incidating apoptosis. There are numerous melanophages (depending on skin type) and disruption of collagen and elastic fibres in papillary dermis.
(Functions of Kindlin-1 in Human Keratinocytes and its Immunolocalization in Human Oral Mucosal Tissues)
DIAGNOSIS OF KS:
KS can be difficult to diagnose at birth because of its considerable overlap with dystrophic epidermolysis bullosa (EB) and EB simplex with mottled pigmentation. The combination of progressive skin atrophy and poikiloderma (both of which tend to be generalized), acral blistering, mucosal inflammation, and varying degrees of photosensitivity, however, is more suggestive of KS. Identification of loss-of-function mutations in the KIND1 (or FERMT1) gene by gene sequencing confirms the diagnosis of KS. Since the identification of the KIND1 gene in 2003, 37 different pathogenic KIND1 mutations have been reported.These comprise 14 nonsense, 13 frameshift, 7 splice site, and 3 large deletion mutations.
(Kindler Syndrome, 2010)
Skin immunofluorescence microscopy using a C-terminal anti-FFH1 antibody typically shows reduced immunolabeling of FFH1 in the basal keratinocyte layer and at the DEJ. This is not a universal finding, however, and variable FFH1 labeling has been observed in several patients with KS.
(Kindlin-1 Is a Phosphoprotein Involved in Regulation of Polarity, Proliferation, and Motility of Epidermal Keratinocytes)
Immunoelectron microscopy demonstrated that kindlin-1 is located in a polarized manner at the basal aspect of keratinocytes facing the basement membrane.
(Kindlin-1 Is a Phosphoprotein Involved in Regulation of Polarity, Proliferation, and Motility of Epidermal Keratinocytes)
MANAGEMENT OF KINDLER SYNDROME
The management of KS is largely symptomatic.The skin in KS is often dry and pruritic and may require frequent topical application of emollients. Photoprotection is advocated because of the development of photosensitivity in KS; many affected individuals typically go red within minutes of sun exposure. There is an increased risk of squamous cell carcinomas and, therefore, repeated screening checks for premalignant keratoses and early malignancy are indicated. Regular dental care is advised because of erosive gingivitis and aggressive periodontitis in KS. Esophageal dilatation may be indicated in patients with dysphagia. In cases of severe esophageal dysfunction, temporary parenteral nutrition may be necessary. Affected individuals with colitislike symptoms may develop iron-deficiency anemia. In some cases, surgical bowel resection may be required for severe colitis. Urethral strictures may also require stenting or surgical intervention. In pregnant women with KS, careful obstetric planning, such as consideration of an elective cesarean section, should be considered because vaginal stenosis may occur in KS.(Kindler Syndrome, 2010)
