Phosphorylation sites in members of the protein kinase A (PKA), PKG, and PKC kinase subfamily are conserved. Thus, the PKB kinase PDK1 may be responsible for the phosphorylation of PKC isotypes. PDK1 phosphorylated the activation loop sites of PKCzeta and PKCdelta in vitro and in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner in vivo in human embryonic kidney (293) cells. All members of the PKC family tested formed complexes with PDK1. PDK1-dependent phosphorylation of PKCdelta in vitro was stimulated by combined PKC and PDK1 activators. The activation loop phosphorylation of PKCdelta in response to serum stimulation of cells was PI 3-kinase-dependent and was enhanced by PDK1 coexpression.

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The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482-789. Talin 482-655, which contains a vinculin-binding site (VBS), folds into a five-helix bundle whereas talin 656-789 is a four-helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482-655 fold. We demonstrate that the talin 482-655 five-helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. Three vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the ␣-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS ␣-helices bind to a hydrophobic cleft in the N-terminal vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX-VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the vinculin Vd1 domain reveals a subtly different mode of vinculin binding.Vinculin (1066 amino acids) is associated with integrin-containing cell-extracellular matrix and cadherin-based cell-cell junctions (1). Electron microscopy images of chicken vinculin show a trilobar head 80 Å in diameter connected to a long flexible "tail" (2), and a recent crystal structure of the full-length vinculin molecule shows a circular five-domain autoinhibited conformation in which the N-terminal head domain (Vd1) 3 forms an extensive interaction with the C-terminal tail domain (Vt) (3). The Vd1 domain contains binding sites for talin (4) and ␣-actinin (5), whereas Vt binds to paxillin (6), F-actin (7), and acidic phospholipids (8). The intramolecular Vd1-Vt interaction regulates vinculin activity by masking the binding sites for talin (9) and ␣-actinin (5) in Vd1, the VASP binding site in the proline-rich domain (10, 11), and the F-actin binding site in Vt (7).Talin (2541 amino acids) is an elongated (60 nm) flexible anti-parallel dimer, with a small globular head connected to an extended rod (2). The talin head contains a FERM domain (residues 86 -400) with binding sites for several ␤-integrin cytodomains (12) as well as the type 1␥ 661 isoform of phosphatidylinositol-4-phosphate 5-kinase (13-15), and the protein-tyrosine kinase FAK (16) both of which are important in focal adhesion dynamics. The talin rod contains a second lower affinity integrin binding site (17, 18), a highly conserved C-terminal actin binding site (residues 2345-2541) (19,20), and also several binding sites for vinculin (19). A yeast two-hybrid assay was used to map three of these vinculin binding sites (VBS1, -2, and -3) to short peptide sequences 25-30 residues in length, ...

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The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into three-dimensional collagen matrices. Cell motility was investigated in vinculin knockout and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knockout cells were 2-fold more motile on two-dimensional collagen-coated substrates compared with wild-type cells, but 3-fold less invasive in 2.4 mg/ml three-dimensional collagen matrices. Vinculin knockout cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced two-dimensional motility but does not explain their reduced three-dimensional invasiveness. Importantly, vinculin-expressing cells adhered more strongly to collagen and generated 3-fold higher traction forces compared with vinculin knockout cells. Moreover, vinculin-expressing cells were able to migrate into dense (5.8 mg/ml) threedimensional collagen matrices that were impenetrable for vinculin knockout cells. These findings suggest that vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.Cell migration is an important and fundamental biomechanical process that plays an essential role in inflammatory diseases, embryonic development, wound healing, and metastasis formation. Current concepts of cell migration have been established in two-dimensional models, but they can explain only partially the migratory behavior in three dimensions. For instance, the migratory capability of cells on two-dimensional substrates depends mainly on adhesion strength, adhesion dynamics, and the dynamics of cytoskeletal remodeling (1, 2), whereas the migratory capability of cells in three-dimensional connective tissue depends also on the steric hindrance of the matrix, matrix degradation by proteolytic enzyme secretion, and the generation of protrusive or contractile forces (1, 3-5). The balance of all these parameters-adhesion strength, cytoskeletal remodeling, matrix degradation, and the generation and transmission of contractile forces-is important for the migration speed in three-dimensional extracellular matrix (ECM) 2 (6). Depending on this balance, a broad variety of invasion strategies between different cell types and even within the same cell type are possible (7).The connection between the ECM and the actomyosin cytoskeleton through integrin-type cell-matrix adhesion receptors is facilitated by the mechano-coupling protein vinculin (8, 9). The effect of vinculin on the migration of cells has previously been investigated using two-dimensional ECM substrates, whe...

There are three conserved phosphorylation sites in protein kinase C (PKC) isotypes that have been termed priming sites and play an important role in PKC function. The requirements and pathways involved in novel (nPKC) phosphorylation have been investigated here. The evidence presented for nPKC␦ shows that there are two independent kinase pathways that act upon the activation loop (Thr-505) and a C-terminal hydrophobic site (Ser-662) and that the phosphorylation of the Ser-662 site is protected from dephosphorylation by the Thr-505 phosphorylation. Both phosphorylations require C1 domain-dependent allosteric activation of PKC. The third site (Ser-643) appears to be an autophosphorylation site. The serum-dependent phosphorylation of the Thr-505 and Ser-662 sites increases nPKC␦ activity up to 80-fold. Phosphorylation at the Ser-662 site is independently controlled by a pathway involving mammalian TOR (mTOR) because the rapamycin-induced block of its phosphorylation is overcome by co-expression of a rapamycin-resistant mutant of mTOR. Consistent with this role of mTOR, amino acid deprivation selectively inhibits the serum-induced phosphorylation of the Ser-662 site in nPKC␦. It is established that nPKC⑀ behaves in a manner similar to nPKC␦ with respect to phosphorylation at its C-terminal hydrophobic site, Ser-729. The results define the regulatory inputs to nPKC␦ and nPKC⑀ and establish these PKC isotypes downstream of mTOR and on an amino acid sensing pathway. The multiple signals integrated in PKC are discussed.