SERINE/THREONINE PHOSPHATASES ARE ...

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(CD11a, CD11b and CD11c) are constitutively phosphorylated (Buyon et al, 1990; Valmu and Gahmberg, 1995). The leukocyte-specific β. 2. -integrins consist of ...
Department of Biosciences and Biotechnology, Division of Biochemistry University of Helsinki, Helsinki, Finland

SERINE/THREONINE PHOSPHATASES ARE INPORTANT FOR REGULAION OF INTEGRIN SIGNALING Ekaterina A. Bryushkova and Carl G. Gahmberg

Introduction Integrins are a large family of heterodimeric transmembrane glycoproteins. They take part in cells interactions with extracellular matrix proteins or with their cellular ligands, like the intercellular adhesion molecules (ICAM) and the vascular adhesion molecule-1 (VCAM-1).

- Two noncovalently associated transmembrane subunits called α (120-170 kDa) and β (90-100 kDa) - Subdivided according to their β chains but some α chains can combine with several β chains - 24 different integrins are present in humans - Play important role in cells adhesion and, as the result, involved in many normal and/or pathological cellular functions: survival, growth, differentiation, migration, inflammatory responses, platelet aggregation, tissue repair and tumor invasion.

The leukocyte-specific β2-integrins consist of four members in humans and mice. They have a common β-chain (β2, CD18) and one of the α-chains: αL(CD11a), αM(CD11b), αX(CD11c) and αD(CD11d). The LFA-1 heterodimer (αLβ2) is primarily expressed in lymphocytes, Mac-1(αMβ2) in granulocytes and both αXβ2 and αDβ2 in monocytes and macrophages (Gahmberg et al, 1997; Luo et al, 2007).

α α α α β2

L

M x

D

Specific studies of β2 family phosphorylation demonstrated that β2-chain is phosphorylated upon cell activation, whereas the α-chains (CD11a, CD11b and CD11c) are constitutively phosphorylated (Buyon et al, 1990; Valmu and Gahmberg, 1995). Previous results suggest that both phorbol ester and CD3 treatment activated PKC. The phosphorylation was strongly augmented by 1.5 µM okadaic acid, an inhibitor of the serine/threonine protein phosphatases PP1 (IC50 = 15-20 nM) and PP2A (IC50 = 0.1 nM). While phorbol ester treatment caused phosphorylation mainly on serine, in okadaic acid-pretreated cells, both phorbol ester treatment as well as CD3 stimulation revealed strong threonine phosphorylation (Valmu and Gahmberg, 1995). This fact indirectly confirms the possibility of serine/threonine phosphatases involvement in the integrins dephosphorylation proses

Object The human T lymphoblastoid cell line clone Jβ2.7, that completely lack cell surface LFA-1 (prepared by chemical mutagenesis and selection as described in Weber et al, 1997) was a gift from N. Hogg and was grown in RPMI 1640 medium supplemented with 10% of FBS, 2mM L-glutamine and 100U/ml penicillin-streptomycin. Jβ2.7 cell lines expressing LFA-1 wt (αL) or the phosphorylation mutant S1140A (αLΔ) were produced by viral transduction (Uotila et al, 2014).

Results I. As far as PP2A is completely inhibited at 1-2 nM of okadaic acid, compared to greater than 1 µM for PP1 (Cohen et al, 1989), we checked out which phosphatases are present in our cell lines. Lysates, 28 mkg of protein per sample PP2A, catalytic subunit, C, 36 kDa PP1, catalytic subunit α, 36 kDa 50

50 Jβ2.7

αL

αL∆

Jβ2.7

αL

αL∆

CLFKSATTTVMN 1

2

3

CLFKSA-pT-TTVMN 1

2

3

- PP1

37

37

25 20

25 20 PP1 antibody (E-9), 1:500, Santa Cruz Biotechnology

II. Role of the PP1 in β2-chain dephosphorylation

PP2AC antibody, 1:1000, Cell Signaling Technology

III. At the presence of R7E4 (the integrin β2 blocking antibody), PP1 seems able to be co-immunoprecipitated with LFA-1 integrins on IB4 antibody, which recognize the CD11a/CD18 heterodimer. These data correlate with the 1 2 1 2 1 2 our previous results, according to which, incubation cells with Jβ2.7 αL αL∆ R7E4 leads to phosphorylation 1 – untreated cells of β2 T758 in αL and αLΔ but not 2 – R7E4 antibody, 20 mkg/ml in Jβ2.7.

+ PP1 45 min, 30°C

Dot blot, anti-CD18/(phospho-Thr758) antibody, 1:1000, Assay Biotech

Sample 1 2 3

Peptide to 1u PP1, mkg 30 15 10

Preliminary in vitro results suggest, that purified PP1 can recognize and dephosphorylate of phosphopeptide (CLFKSApTTTVMN) that mimic the phosphorylation consensus sequence in the cytoplasmic part of the β2-chain. The research is supported by CIMO Fellowship