Modulation of protein 4.1 binding to inside-out membrane vesicles by phosphorylation

Abstract

The effect of phosphorylation on the binding of protein 4.1 to erythrocyte inside-out vesicles was investigated. Protein 4.1 was phosphorylated with casein kinase A, protein kinase C, and cAMP-dependent protein kinase. An analysis of the phosphopeptides generated by alpha-chymotryptic and tryptic digestion indicates these kinases phosphorylate similar as well as distinct domains within protein 4.1. All three enzymes catalyze the phosphorylation to varying degrees of the 46-, 16-, and 8-10-kDa fragments derived from limited chymotryptic cleavage. In addition, casein kinase A phosphorylates a 24-kDa domain, whereas protein kinase C phosphorylates a 30-kDa domain. Protein 4.1 phosphorylated by casein kinase A and protein kinase C, but not cAMP-dependent protein kinase, exhibits a reduced binding to KI-extracted inside-out vesicles. On the other hand, phosphorylation of inside-out vesicles by casein kinase A does not affect their ability to bind protein 4.1. The inside-out vesicles, however, inhibit the phosphorylation of protein 4.1 by casein kinase A and protein kinase C, but not by cAMP-dependent protein kinase. These results suggest that casein kinase A and protein kinase C may modulate the binding of protein 4.1 to the membrane by phosphorylation of specific domains of the cytoskeletal protein. Since the 30-kDa domain has been suggested as a membrane-binding site, that phosphorylation by protein kinase C reduces the binding of protein 4.1 to inside-out vesicles is perhaps not surprising. On the other hand, the role of the casein kinase A substrate 24-kDa domain in membrane binding has not been established and needs to be examined.(ABSTRACT TRUNCATED AT 250 WORDS)

A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity

“…This is consistent with the fact that it directly interacts with aPKC (Fig. 1 D) and with previous studies showing that the FA domain of other FERM family members acts as an important phosphorylation-dependent regulatory element (Danilov et al, 1990; Chao and Tao, 1991; Manno et al, 2005; Baines, 2006; Nakajima and Tanoue, 2011). Therefore, we focused our analysis on the FA domain of Yrt, which contains 12 serine/threonine (S/T) residues (Fig.…”

Reversible erythrocyte skeleton destabilization is modulated by beta-spectrin phosphorylation in childhood leukemia

“…Several observations based on studies in vitro strongly suggest that phosphorylation may modify interactions between proteins, leading in all cases to a reduced affinity. [9][10][11][12][13][14][15][16][17][18] Our study shows an ellipto-poikilocytosis, an abnormal spectrin dimer percentage and an increased ␤-spectrin phosphorylation in 10 patients with leukemia at diagnosis with their normalization during the remission phase. Although in vitro studies showed that the phosphorylation did not affect the dimer-tetramer equilibrium of spectrin 41,42 and the erythrocyte shape, 43 our results show a different pattern in vivo.…”

“…8 Evidence shows that the shape and the deformability of the red blood cell membrane are very sensitive to the state of phosphorylation. [9][10][11][12][13][14][15][16][17][18] In particular, increased phosphorylation of ␤-spectrin decreases membrane mechanical stability. 19 Several acquired abnormalities of erythrocytes and platelets have been described in leukemia and myelodysplastic syndromes.…”

Regulation of the Glycophorin C-Protein 4.1 Membrane-to-Skeleton Bridge and Evaluation of Its Contribution to Erythrocyte Membrane Stability

“…A variety of observations in the literature, stemming primarily from studies on purified membrane components, have suggested that the glycophorin C-protein 4.1 bridge to the membrane skeleton might be physiologically regulated (22,24,(32)(33)(34). However, except for the impact of protein kinase C phosphorylation on this bridging function (35), none of the potential regulatory pathways has been validated in intact erythrocytes.…”