Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.

Schook, William; Puszkin, Saul

doi:10.1073/pnas.82.23.8039

Cited by 36 publications

(45 citation statements)

References 28 publications

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“…Substitution of an acidic residue at the third position downstream of the serine in GGA2 allowed the molecule to be phosphorylated by CK2. Although the actual kinase that phosphorylates GGA1 and GGA3 in cells has yet to be identified, it should be noted that a CK2-type enzyme has been shown to be associated with clathrin-coated vesicles (16,(21)(22)(23)(24). In preliminary experiments, we found that an activity associated with purified clathrin-coated vesicles phosphorylates the GGA1 peptide.…”

Section: Discussionmentioning

confidence: 99%

Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif

Doray

Bruns²,

Ghosh³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

111

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The GGAs (Golgi-localizing, ␥-adaptin ear homology domain, ARFbinding proteins) are a family of proteins implicated in protein trafficking from the Golgi to endosomes͞lysosomes. These proteins have modular structures with an N-terminal VHS (VPS-27, Hrs, and STAM) domain followed by a GAT (GGA and TOM1) domain, a connecting hinge segment, and a C-terminal GAE (␥-adaptin ear) domain. Isolated VHS domains have been shown to bind specifically to acidic cluster (AC)-dileucine motifs present in the cytoplasmic tails of the mannose 6-phosphate receptors. Here we report that full-length cytoplasmic GGA1 and GGA3 but not GGA2 bind the cation-independent mannose 6-phosphate receptor very poorly because of autoinhibition. This inhibition is caused by the binding of an AC-LL sequence present in the hinge segment to the ligand-binding site in the VHS domain. The inhibition depends on the phosphorylation of a serine located three residues upstream of the AC-LL motif. The serine is phosphorylated by casein kinase 2 in in vitro assays. Substitution of the GGA1 inhibitory sequence into the analogous location in GGA2, which lacks the AC-LL motif, results in autoinhibition of the latter protein. These data indicate that the activity of GGA1 and GGA3 is regulated by cycles of phosphorylation͞dephosphorylation.

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Section: Discussionmentioning

confidence: 99%

Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif

Doray

Bruns²,

Ghosh³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

111

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“…AtT-20#1 cells stably expressing GAIP were prepared as described previously (25,27) and labeled for 4 h with either 100 Ci͞ml Easytag or 1 mCi͞ml [ 32 P]orthophosphate. 35 S labeling was performed in Cys-and Met-free DMEM supplemented with 4% dialysed FCS. 32 P labeling was carried out in phosphate-free DMEM supplemented with 4% FCS.…”

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confidence: 99%

Membrane-associated GAIP is a phosphoprotein and can be phosphorylated by clathrin-coated vesicles

Elenko

Wan

Thomas

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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GAIP (G ␣ interacting protein) is a member of the RGS (regulators of G protein signaling) family and accelerates the turnover of GTP bound to G␣i, G␣q, and G␣13. There are two pools of GAIP-a soluble and a membrane-anchored pool. The membrane-anchored pool is found on clathrin-coated vesicles (CCVs) and pits in rat liver and AtT-20 pituitary cells. By treatment of a GAIP-enriched rat liver fraction with alkaline phosphatase, we found that membranebound GAIP is phosphorylated. By immunoprecipitation carried out on [ 32 P]orthophosphate-labeled AtT-20 pituitary cells stably expressing GAIP, 32 P-labeling was associated exclusively with the membrane pool of GAIP. Phosphoamino acid analysis revealed that phosphorylation of GAIP occurred largely on serine residues. Recombinant GAIP could be phosphorylated at its N terminus with purified casein kinase 2 (CK2). It could also be phosphorylated by isolated CCVs in vitro. Phosphorylation was Mn 2؉ -dependent, using both purified CK2 and CCVs. Ser-24 was identified as one of the phosphorylation sites. Our results establish that GAIP is phosphorylated and that only the membrane pool is phosphorylated, suggesting that GAIP can be regulated by phosphorylation events taking place at the level of clathrin-coated pits and vesicles.G proteins ͉ regulator of G protein signaling ͉ casein kinase 2 S ignal transduction components and G-protein coupled receptor (GPCR) signaling pathways in particular undergo phosphorylation and dephosphorylation events that control their effects on multiple physiological processes (1). For example, GPCR are phosphorylated by G protein-coupled receptor kinases (GRKs) (2, 3), and several G proteins undergo phosphorylation in vivo on their ␣ (4-6) and ␥ (7) subunits, which may regulate their membrane targeting (8) and͞or interaction with partners (9)(10)(11)(12) Recently, a family of more than 20 members, the RGS proteins (for regulators of G protein signaling), have been identified that act as GTPase activating proteins (GAPs) for G␣ subunits (for reviews see refs. 13 and 14). We localized one member of the RGS family, RGS-GAIP (G ␣ interacting protein), by immunocytochemistry and found it to be associated with clathrincoated pits and vesicles (15,16) and further demonstrated that CCVs isolated from rat liver possess GAP activity (16).Clathrin-coated vesicles (CCVs) are known to play a role in G-protein coupled receptor-(2) and receptor-mediated endocytosis at the plasma membrane and in sorting of lysosomal enzymes in the trans-Golgi network (TGN) (17)(18)(19). Formation of CCVs is known to be tightly regulated by phosphorylation and dephosphorylation of adapters and coat proteins (20)(21)(22)(23)(24).GAIP contains nine putative phosphorylation sites-two for protein kinase C (PKC) and seven for casein kinase 2 (CK2) (25). These consensus sites are distributed over all three domains of GAIP: (i) its highly conserved RGS domain (amino acids 86-205) required for GAP activity (26), (ii) its N terminus (amino acids 1-85) most likely responsible for membrane ...

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“…Morphological alterations of the cell surface and cytoskeleton are among the earliest manifestations of the src gene product (Ambros et al, 1975; Boschek et al, 1981). Among the cell surface changes, it has been observed that a number of receptors, including those for epidermal growth factor and thrombin, are cleared from the surface by endocytic events that may be altered by pp60 ~-s~ (Decker, 1983;Cooper et al, 1983;Zetter et al, 1977).Endocytosis often proceeds via coated pits and coated vesicles (for review see Pastan and Willingham, 1985), structures whose coats are known to include several phosphoproteins, among them the assembly factor polypeptides (Pauloin et al, 1982;Campbell et al, 1984;Keen et al, 1987) and the clathrin light chains (Usami et al, 1985;Schook and Puszkin, 1985;Bar-Zvi and Branton, 1986 phorylation both in vivo and in vitro (Keen and Black, 1986; Bar-Zyi et al, 1988b), phosphorylation of clathrin heavy chain, the major component of coated vesicles, has not been observed in normal cells. Here we show that transformation of secondary chick embryo fibroblasts (CEF) by RSV induces the phosphorylation of clathrin heavy chains on tyrosine and serine residues.…”

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confidence: 99%

“…Endocytosis often proceeds via coated pits and coated vesicles (for review see Pastan and Willingham, 1985), structures whose coats are known to include several phosphoproteins, among them the assembly factor polypeptides (Pauloin et al, 1982;Campbell et al, 1984;Keen et al, 1987) and the clathrin light chains (Usami et al, 1985;Schook and Puszkin, 1985;Bar-Zvi and Branton, 1986 phorylation both in vivo and in vitro (Keen and Black, 1986; Bar-Zyi et al, 1988b), phosphorylation of clathrin heavy chain, the major component of coated vesicles, has not been observed in normal cells. Here we show that transformation of secondary chick embryo fibroblasts (CEF) by RSV induces the phosphorylation of clathrin heavy chains on tyrosine and serine residues.…”

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confidence: 99%

Transformation by Rous sarcoma virus induces clathrin heavy chain phosphorylation.

Martı́n-Pérez

Bar-Zvi

Branton

et al. 1989

The Journal of Cell Biology

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Abstract. We have shown that the heavy chain of clathrin is phosphorylated in chicken embryo fibroblast cells transformed by Rous sarcoma virus, but not in normal cells. Approximately 1 mol of phosphate is bound for every 5 mol of heavy chain in the maximally phosphorylated transformed cells. Two-thirds of the phosphate is on serine and one-third on tyrosine residues. Clathrin heavy chain is a substrate for pp60 .... in vitro. Cleveland analysis of the in vivo and in vitro clathrin heavy chain phosphopeptides, generated by protease V8 digestion, show labeled proteolytic fragments of similar molecular weight, suggesting that pp60 V-sfc could be directly responsible for the in vivo phosphorylation of clathrin. Phosphate is equally incorporated into clathrin in both the unassembled and the assembled clathrin pools, whereas [35S]methionine is preferentially incorporated into the assembled pool. In normal cells, clathrin visualized by immunofluorescent staining appears in a punctate pattern along the membrane surface and concentrated around the nucleus; in transformed cells the perinuclear staining is completely absent. The phosphorylation of clathrin heavy chain in transformed cells may be linked to previously observed transformation-dependent alterations in receptor-mediated endocytosis of ligands such as EGF and thrombin.T HE search for the underlying molecular mechanisms that explain transformation by Rous sarcoma virus (RSV) ~ has resulted in the identification of many substrates phosphorylated by the tyrosine kinase, pp60v-~% as well as numerous substrates phosphorylated by other kinases whose activities are indirectly regulated by RSV (for reviews see Cooper, 1985, 1986;Jove and Hanafusa, 1987). Morphological alterations of the cell surface and cytoskeleton are among the earliest manifestations of the src gene product (Ambros et al., 1975; Boschek et al., 1981). Among the cell surface changes, it has been observed that a number of receptors, including those for epidermal growth factor and thrombin, are cleared from the surface by endocytic events that may be altered by pp60 ~-s~ (Decker, 1983;Cooper et al., 1983;Zetter et al., 1977).Endocytosis often proceeds via coated pits and coated vesicles (for review see Pastan and Willingham, 1985), structures whose coats are known to include several phosphoproteins, among them the assembly factor polypeptides (Pauloin et al., 1982;Campbell et al., 1984;Keen et al., 1987) and the clathrin light chains (Usami et al., 1985;Schook and Puszkin, 1985;Bar-Zvi and Branton, 1986 phorylation both in vivo and in vitro (Keen and Black, 1986; Bar-Zyi et al., 1988b), phosphorylation of clathrin heavy chain, the major component of coated vesicles, has not been observed in normal cells. Here we show that transformation of secondary chick embryo fibroblasts (CEF) by RSV induces the phosphorylation of clathrin heavy chains on tyrosine and serine residues. The effect is paralleled by a partial cellular redistribution of clathrin. Materials and Methods Cell Culture and LabelingCEF were...

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Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.

Cited by 36 publications

References 28 publications

Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif

Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif

Membrane-associated GAIP is a phosphoprotein and can be phosphorylated by clathrin-coated vesicles

Transformation by Rous sarcoma virus induces clathrin heavy chain phosphorylation.

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