Ganglioside Loss Promotes Survival Primarily by Activating Integrin-Linked Kinase/Akt Without Phosphoinositide 3-OH Kinase Signaling

Abstract

Keratinocyte gangliosides influence cellular functions, including proliferation, adhesion, migration, and differentiation. The effects of endogenous depletion of membrane gangliosides by gene transfection of a human ganglioside-specific sialidase on cell survival were investigated. Ganglioside depletion promotes survival of the human keratinocyte-derived SCC12 cell line through upregulated phosphorylation of beta1 integrin, and increased phosphorylation and activity of integrin-linked kinase, protein kinase B/Akt, and Bad, with resultant inhibition of caspase-9 activation. Ganglioside deficiency also increases expression of cyclins D1 and E, promoting cell cycle progression from G1 phase to S phase. Inhibition of either protein kinase B/Akt or integrin-linked kinase activity renders the ganglioside-deficient cells susceptible to triggers of apoptosis. Both serine-473 and threonine-308 sites of protein kinase B/Akt show increased phosphorylation in ganglioside-deficient cells, but the cell survival correlates with increased phosphorylation of the serine-473 site of Akt, not with increased phosphorylation of the threonine-308 site. Consistently, blockade of ganglioside GT1b function activates integrin-linked kinase and only the serine-473 site of protein kinase B/Akt. In contrast, antibody-induced blockade of GM3 function increases only threonine-308 phosphorylation of ganglioside-deficient cells. Whereas blockade of phosphoinositide 3-OH kinase function suppresses threonine-308 phosphorylation, it neither inhibits serine-473 phosphorylation nor triggers apoptosis. These data suggest that ganglioside depletion modulates cell survival primarily through protein kinase B/Akt stimulation by a pathway that does not require phosphoinositide 3-OH kinase and epidermal growth factor receptor signaling.

Activation of the protein kinase B pathway by the HPV-16 E7 oncoprotein occurs through a mechanism involving interaction with PP2A

“…The proliferative pathway appears to be switched off and the differentiation pathway switched on in keratinocytes that have divided up from the basal layer; both effects mediated by their loss of contact with the basal membrane and consequent loss of interaction between integrins and laminins 5/10/11 (Fujisaki and Hattori, 2002;Pouliot et al, 2002). The loss of integrin/laminin interaction inactivates PI3K and subsequently PKB/Akt, and it is noteworthy that one of the kinases that activate Akt by phosphorylation on ser473 is ILK (Delcommene et al, 1998), and it has also been shown that ILK can phosphorylate ser473 of PKB/ Akt in the absence of PI3K-mediated phosphorylation of thr308 (Sun et al, 2002). In the context of these observations, the finding that E7 proteins may increase the PI3K-independent phosphorylation of PKB/Akt, thereby inhibiting apoptosis during viral DNA amplification in the suprabasal layers of HPV-infected host keratinocytes is consistent with our understanding of the HPV life cycle, given that loss of contact with the basal membrane would tend to switch off host cell DNA synthesis as cells begin their normal differentiation schedule.…”

Ganglioside GM3 Blocks the Activation of Epidermal Growth Factor Receptor Induced by Integrin at Specific Tyrosine Sites

“…SCC12 cells were stably transfected with human plasma membrane ganglioside-specific sialidase cDNA (GenBank TM accession number AB008185, courtesy of Dr. T. Miyagi, Tokyo, Japan) (32) in a pcDNA3 vector using LipofectAMINE reagent (18,22). Gene and protein expression in the resultant SSIA cells were demonstrated by Northern blot and sialidase activity measurements.…”

“…Gene and protein expression in the resultant SSIA cells were demonstrated by Northern blot and sialidase activity measurements. Ganglioside depletion was shown by thin layer chromatography (TLC) immunostaining (18,20,22). Four SSIA cell lines (SSIA3, SSIA6, SSIA12, and SSIA25) and 2 mock-transfected pcDNA cell lines were studied.…”

Role of the integrin-linked kinase/PINCH1/alpha-parvin complex in cardiac myocyte hypertrophy

“…14 ILK kinase activity is stimulated by growth factors and by attachment of cells to the extracellular matrix, namely to FN, [14][15][16] and is localized to the cytoskeletal fraction. 14 Inhibition of the formation of the IPAP1 complex or of ILK activity in epithelial cells has resulted in decreased cell spreading and motility, 8,14,17 growth suppression 18 and apoptosis, [19][20][21][22] and therefore it has been proposed as a potential target for cancer treatment. 23 Cardiac hypertrophy reproduces some of the molecular features of the neoplastic phenotype in other organs and has been associated with remodeling of the extracellular matrix and increased expression and cytoskeletal association of fibronectin (FN), vitronectin and paxillin.…”