Phosphorylation of threonine 1736 in the C-terminal tail of integrin ß4 contributes to hemidesmosome disassembly.

During wound healing, hemidesmome disassembly enables keratinocyte migration and proliferation. Hemidesmosome dynamics are altered downstream of epidermal growth factor (EGF) receptor activation, following the phosphorylation of integrin ß4 residues S1356 and S1364, which reduces the interaction with plectin; however, this event is insufficient to drive complete hemidesmome disassembly. In the studies reported here, we used a fluorescence resonance energy transfer-based assay to demonstrate that the connecting segment and carboxy-terminal tail of the ß4 cytoplasmic domain interact, which facilitates the formation of a binding platform for plectin. In addition, analysis of a ß4 mutant containing a phosphomimicking aspartic acid residue at T1736 in the C-tail suggests that phosphorylation of this residue regulates the interaction with the plectin plakin domain. The aspartic acid mutation of ß4 T1736 impaired hemidesmosome formation in junctional epidermolysis associated with pyloric atresia/ß4 keratinocytes. Furthermore, we show that T1736 is phosphorylated downstream of protein kinase C and EGF receptor activation and is a substrate for protein kinase D1 in vitro and in cells, which requires its translocation to the plasma membrane and subsequent activation. In conclusion, we identify T1736 as a novel phosphorylation site that contributes to the regulation of hemidesmome disassembly, a dynamically regulated process involving the concerted phosphorylation of multiple ß4 residues.

Nesprin-3 augments peripheral nuclear localization of intermediate filaments in zebrafish.

The outer nuclear membrane protein nesprin-3 binds the cytoskeletal linker protein plectin, which are proposed to anchor the intermediate filaments to the nuclear envelope. To investigate the function of nesprin-3 in vivo, we used the zebrafish as a vertebrate model system. Zebrafish nesprin-3 is expressed at the nuclear envelope of epidermal and skeletal muscle cells during development. Unexpectedly, loss of nesprin-3 did not affect embryonic development, viability or fertility. However, nesprin-3-deficient zebrafish embryos showed a reduced concentration of intermediate filaments around the nucleus. Additional analysis revealed the presence of two nesprin-3 isoforms in zebrafish, nesprin-3α and nesprin-3ß. Nesprin-3ß is only expressed during early development and lacks seven amino acids in its first spectrin repeat that are crucial for plectin binding and recruitment to the nuclear envelope. These seven amino acids are highly conserved and we showed that residues R43 and L44 within this motif are required for plectin binding. Furthermore, several residues in the actin-binding domain of plectin that are crucial for binding to the integrin ß4 subunit are also important for the binding to nesprin-3α, indicating partial overlapping binding sequences for nesprin-3α and integrin ß4. All this shows that nesprin-3 is dispensable for normal development in zebrafish, but important for mediating the association of the intermediate filament system with the nucleus in vivo.

Requirements for the localization of nesprin-3 at the nuclear envelope and its interaction with plectin.

The outer nuclear membrane proteins nesprin-1 and nesprin-2 are retained at the nuclear envelope through an interaction of their klarsicht/ANC-1/syne homology (KASH) domain with Sun proteins present at the inner nuclear membrane. We investigated the requirements for the localization of nesprin-3alpha at the outer nuclear membrane and show that the mechanism by which its localization is mediated is similar to that reported for the localization of nesprin-1 and nesprin-2: the last four amino acids of the nesprin-3alpha KASH domain are essential for its interaction with Sun1 and Sun2. Moreover, deletion of these amino acids or knockdown of the Sun proteins results in a redistribution of nesprin-3alpha away from the nuclear envelope and into the endoplasmic reticulum (ER), where it becomes colocalized with the cytoskeletal crosslinker protein plectin. Both nesprin-3alpha and plectin can form dimers, and dimerization of plectin is required for its interaction with nesprin-3alpha at the nuclear envelope, which is mediated by its N-terminal actin-binding domain. Additionally, overexpression of the plectin actin-binding domain stabilizes the actin cytoskeleton and prevents the recruitment of endogenous plectin to the nuclear envelope. Our studies support a model in which the actin cytoskeleton influences the binding of plectin dimers to dimers of nesprin-3alpha, which in turn are retained at the nuclear envelope through an interaction with Sun proteins.

Serine phosphorylation of the integrin beta4 subunit is necessary for epidermal growth factor receptor induced hemidesmosome disruption.

Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of alpha6beta4 to plectin plays a central role in their assembly. We have defined three regions on beta4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of beta4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of beta4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the beta4 subunit.

The regulation of MacMARCKS expression by integrin beta3.

Integrin-mediated adhesion regulates multiple signaling pathways. Our group previously showed that ectopic expression of different integrin beta-subunits in the neuroepithelial cell line GE11, has distinct effects on cell morphology, actin cytoskeletal organization, and on focal contact distribution. In this report we have investigated changes in gene transcription levels resulting from overexpression of the integrin beta3 subunit. We found that beta3 overexpression leads to the transcriptional downregulation of MARCKS related protein (MRP) resulting in a decreased expression of the MRP protein. Furthermore, we show that the Ras/MAPK pathway controls the basal level of MRP expression but beta3 overexpression bypasses this pathway downstream of ERK to downregulate MRP. Further studies indicate that a region of the cytoplasmic tail of beta3 containing part of the NITY motif is responsible for increased cell spreading and MRP downregulation. However, MRP overexpression failed to inhibit the beta3-induced increase in cell spreading while the knock down of MRP expression in GE11 cells did not increase cell spreading. We suggest that the downregulation of MRP by beta3 is not required for increased cell spreading but instead that MRP downregulation is a secondary effect of increased cell spreading.

Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin.

Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)-1 and -2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin alpha6beta4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton.

Specificity of binding of the plectin actin-binding domain to beta4 integrin.

Plectin is a major component of the cytoskeleton and links the intermediate filament system to hemidesmosomes by binding to the integrin beta4 subunit. Previously, a binding site for beta4 was mapped on the actin-binding domain (ABD) of plectin and binding of beta4 and F-actin to plectin was shown to be mutually exclusive. Here we show that only the ABDs of plectin and dystonin bind to beta4, whereas those of other actin-binding proteins do not. Mutations of the ABD of plectin-1C show that Q131, R138, and N149 are critical for tight binding of the ABD to beta4. These residues form a small cavity, occupied by a well-ordered water molecule in the crystal structure. The beta4 binding pocket partly overlaps with the actin-binding sequence 2 (ABS2), previously shown to be essential for actin binding. Therefore, steric interference may render binding of beta4 and F-actin to plectin mutually exclusive. Finally, we provide evidence indicating that the residues preceding the ABD in plectin-1A and -1C, although unable to mediate binding to beta4 themselves, modulate the binding activity of the ABD for beta4. These studies demonstrate the unique property of the plectin-ABD to bind to both F-actin and beta4, and explain why several other ABD-containing proteins that are expressed in basal keratinocytes are not recruited into hemidesmosomes.

Knock-in of integrin beta 1D affects primary but not secondary myogenesis in mice.

Integrins are extracellular matrix receptors composed of alpha and beta subunits involved in cell adhesion, migration and signal transduction. The beta1 subunit has two isoforms, beta 1A ubiquitously expressed and beta 1D restricted to striated muscle. They are not functionally equivalent. Replacement of beta 1A by beta 1D (beta 1D knock-in) in the mouse leads to midgestation lethality on a 50% Ola/50% FVB background [Baudoin, C., Goumans, M. J., Mummery, C. and Sonnenberg, A. (1998). Genes Dev. 12, 1202-1216]. We crossed the beta 1D knock-in line into a less penetrant genetic background. This led to an attenuation of the midgestation lethality and revealed a second period of lethality around birth. Midgestation death was apparently not caused by failure in cell migration, but rather by abnormal placentation. The beta 1D knock-in embryos that survived midgestation developed until birth, but exhibited severely reduced skeletal muscle mass. Quantification of myotube numbers showed that substitution of beta 1A with beta 1D impairs primary myogenesis with no direct effect on secondary myogenesis. Furthermore, long-term primary myotube survival was affected in beta 1D knock-in embryos. Finally, overexpression of beta 1D in C2C12 cells impaired myotube formation while overexpression of beta 1A primarily affected myotube maturation. Together these results demonstrate for the first time distinct roles for beta1 integrins in primary versus secondary myogenesis and that the beta 1A and beta 1D variants are not functionally equivalent in this process.

Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits.

Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin beta1A and beta1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (DeltaH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin beta subunits. The mere presence of the high-affinity binding site for beta1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.