Specific Residues within the α2 Integrin Subunit Cytoplasmic Domain Regulate Migration and Cell Cycle Progression via Distinct MAPK Pathways

Paul A Klekotka,Samuel A Santoro,Haochuan Wang,Mary M Zutter

doi:10.1074/jbc.m101921200

Paul A Klekotka, Samuel A Santoro + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m101921200

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Abstract

The alpha(2) integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the alpha(2) integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression.

Highlights

Tactic migration integrin ligation provides the adhesion necessary for traction on the extracellular matrix and activates intracellular signaling molecules such as Rac, Cdc42, and p38 MAPK [12, 13]
Clones of the ␣2 integrin cytoplasmic domain truncations, 1146 and 1139, migrated in response to epidermal growth factor (EGF) on type I collagen matrices in a manner comparable to cells expressing the fulllength ␣2 integrin cytoplasmic domain (X2C2). Both the 1146 and 1139 transfectants were able to enter S-phase, as measured by bromodeoxyuridine (BrdUrd) incorporation, following adhesion to type I collagen in the presence of insulin as effectively as the X2C2 transfectants (Fig. 2B). These results indicate that the seven amino acids following the GFFKR motif of the ␣2 integrin cytoplasmic domain were sufficient to support EGF-stimulated chemotactic migration as well as insulin-dependent entry into S-phase in mammary epithelial cells adherent to type I collagen
The methionine at position 1137 is required for both the sustained phosphorylation of the p38 MAPK leading to ␣2 cytoplasmic domain-dependent, EGFstimulated migration and sustained phosphorylation of the extracellular signal-regulated kinase (ERK) MAPK, leading to up-regulation of cyclin E and cdk2 levels, and entry into S-phase

Summary

MAPK Pathways*

Mutation of the lysine at position 1136 to alanine severely inhibited the ability of the mammary epithelial cells to enter S-phase, whereas it had no effect on the ability of the cells to migrate in response to EGF on a type I collagen matrix. These results confirmed our previous findings that phosphorylation of p38 MAPK downstream of the ␣2 integrin cytoplasmic domain was required for EGF-stimulated chemotactic migration and that up-regulation of cyclin E and cyclin dependent kinase 2 (cdk2) was required for entry into S-phase

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION