Site-Directed Perturbation of Protein Kinase C- Integrin Interaction Blocks Carcinoma Cell Chemotaxis

Abstract

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C␣ (PKC␣) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC␣ V3 hinge domain. This motif is also required for a direct association between PKC␣ and ␤1 integrin. Efficient binding of ␤1 integrin to PKC␣ requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of ␤1 integrin was shown to block both PKC␣-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC␣ and ␤1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.An early critical step within the metastatic cascade involves cell polarization and directed cell movement. The mechanisms controlling these events, however, are poorly understood. Cell polarization signals can be triggered by the binding of cells to extracellular matrix (ECM) proteins, cytokines or chemoattractants, or ligands that are expressed on the surface of target tissues or vessels. Sensing of these extracellular signals results in a redistribution and trafficking of membrane receptors (1, 19) and/or intracellular signaling proteins, as well as the localized formation of protein complexes which can be stable or transient.Members of the integrin receptor family are vital in mediating cell matrix adhesion and have been implicated in providing the persistence and directionality for cell motility, as in wound healing (7). Specifically, a number of studies have demonstrated that the ␤-subunit cytoplasmic domains are involved in the control of cell migration. Thus, the tyrosine residues of the two NPXY motifs in ␤1 are important for directed cell migration through integrin substrate-coated filters in response to growth factor chemotactic gradients (22,23). In fact, when expressed as chimeric receptors connected to heterologous extracellular domains, such as the interleukin-2 receptor (IL-2R) tac subunit, the ␤1 integrin cytoplasmic tail acts as a dominant negative inhibitor of endogenous integrin function (4). Moreover, inhibition of integrin-ECM interactions by GRGDS peptides or inhibitory antibodies was also shown to abolish the persistence or directionality of neural crest cell movement (7).Protein kinase C (PKC) may be seen as part of the molecular machinery that deciphers...

Pentoxifylline modulates cell surface integrin expression and integrin mediated adhesion of B16F10 cells to extracellular matrix components

“…MBP 4-14 has been shown to be a specific substrate for PKC that reacts broadly with most isoforms of PKC, although its reactivity with PKC-d and -z is relatively weak. 18,22 Cells were also treated with Calphostin C to determine the specificity of the assay. Calphostin C has been shown to be a specific inhibitor of PKC (IC 50 = 50 nM) and inhibits other kinases at concentrations above 5 mM.…”

“…PKC activity was determined using the method of Yasuda et al 21 with minor modifications. 10 mg of the cell lysate was incubated for 20 min at 30°C in 40 ml of reaction containing 20 mM Tris-HCl (pH 7.5), 5 mM Mg(OAc)2, 0.2 mM CaCl2, 50 mM ATP, 2 mCi [g -32P ]ATP, 0.01 mg/ml Leupeptin and 50 mg/ml MBP4-14 as a substrate 18 with or without 25 mg/ml phosphatidylserine and 50 ng/ml TPA. The reaction was stopped by the addition of 300 mM orthophsophoric acid and 20 ml of the sample was spotted onto P81 phosphocellulose discs.…”

“…So in this study we looked at the effect of PTX on integrin expression on B16-F10 cells and integrin mediated adhesion of B16-F10 melanoma cells. Protein kinase C activity has been shown to be important in regulation of integrin expression, localization 17,18 and activity. 19 Hence we decided to check whether PTX could inhibit PKC activity in our system.…”

The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase Cα

“…The two CH domains at the filamin N terminus did not contribute to PKC binding (Fig. 3B, compare [N1-11] to [1][2][3][4][5][6][7][8][9][10][11]). This overlay assay, however, did not allow a more detailed analysis of the N-terminal binding site for PKC in filamin, since constructs including partial ␤-sheets displayed highly variable binding (data not shown).…”

“…7 and 8). The regulatory role of PKC is based on different mechanisms: PKC directly interacts with transmembrane receptors like ␤ 1 -integrins (9), and integrin co-receptors, syndecan 4 (10) and tetraspanins (11). In addition, co-localization of some PKC isotypes with cytosolic components of the microfilament system was observed (3,12), and a number of actin-binding and modulating proteins, e.g.…”