p24 is a thyroid protein (Mr 24,000) identified by two‐dimensional gel electrophoresis on the basis that its synthesis and phosphorylation are up‐regulated by thyrotropin and cyclic AMP agonists. p24 cDNA was cloned from a lambda gt11 cDNA library using a polyclonal antibody raised against the protein recovered from a Western blot spot. The encoded polypeptide (189 residues) displays a putative target‐site for phosphorylation by cyclic AMP‐dependent protein kinase and belongs to the superfamily of proteins binding Ca2+ through ‘EF hand’ domains. It presents four such domains of which two agree closely with the consensus. The ability of p24 to bind Ca2+ has been directly confirmed on Western blots. p24 was detected in many tissues including the salivary glands, the lung and the brain. The ubiquitous nature of p24, together with its regulatory and sequence characteristics suggest that it constitutes an important target common to the cyclic AMP and Ca2+‐phosphatidylinositol cascades.

In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.

Using separation of total cellular proteins by two dimensional (2-D) gel electrophoresis (isoelectric focusing/SDS-PAGE) we have characterized two regulated proteins, p21 and p19, in dog thyroid cells. We have used the same 2-D gel technique to purify these proteins before their trypsin cleavage and partial sequencing. Three peptides were sequenced in the case of p19 and two peptides in the case of p21. The Swiss-Prot protein sequence database revealed that p19 was identical to destrin/ADF (actin depolymerizing factor) and p21 to cofilin, two closely related and widely distributed actin-binding proteins. This was further verified by cross-reactivity with specific antibodies against brain cofilin and chicken ADF. We have demonstrated, using 2-D gel electrophoresis with a nonequilibrium pH gradient in the first dimension (nonequilibrium pH gradient in the first dimension (nonequilibrium pH gradient electrophoresis/SDS-PAGE) that, in the thyroid cell, cofilin and destrin/ADF were present, under control conditions, in two forms: a phosphorylated and an unphosphorylated one. Thyrotropin (TSH), through cyclic AMP, provoked a very rapid dephosphorylation of these two proteins, which was already maximal after 20 min of action, whereas their dephosphorylation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) was slower. This suggests that dephosphorylation of cofilin and destrin/ADF by TSH could be implicated in the disruption of actin-containing stress fibers and in the reorganization of microfilaments induced by this hormone. Epidermal growth factor, which does not induce acute morphological changes in thyroid cells, did not affect the state of phosphorylation of cofilin and destrin/ADF except for a delayed decrease (after 24 h) of destrin/ADF phosphorylation. A 10% dimethyl sulfoxide treatment of thyroid cells also induced rapid dephosphorylation of destrin and cofilin. This was accompanied by a reorganization of actin microfilaments that clearly resembles the one induced by TSH and by the appearance of intranuclear cofilin-containing rods. However, these rod structures were not observed in response to TSH, forskolin, or TPA, suggesting that dephosphorylation of cofilin correlates with the reorganization of actin microfilaments but not with the nuclear transport of cofilin. We propose that the dephosphorylation of destrin and cofilin could be involved in the TSH-stimulated macropinocytic activity, a key process in thyroid hormone secretion.

In a wide variety of cell types D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 1 and 1,2-diacylglycerol are generated from phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) by receptor-mediated activation of phospholipase C (for review, see Refs. 1 and 2). Ins(1,4,5)P 3 mobilizes intracellular calcium from internal stores generating calcium signals to control many cellular processes: smooth muscle contraction, secretion, sensory perception, neuronal signaling, and cell growth (2). Ins(1,4,5)P 3 can be dephosphorylated by a 5-phosphatase to produce Ins(1,4)P 2 and phosphorylated by a 3-kinase to produce Ins(1,3,4,5)P 4 (3-5) (for review, see Refs. 2, 6, and 7). Some evidence supports a role for Ins(1,3,4,5)P 4 in the regulation of intracellular free calcium concentration in concert with Ins(1,4,5)P 3 (for review, see Refs. 7 and 8). Recently, a specific Ins(1,3,4,5)P 4 -binding protein has been isolated and identified as a member of the GAP1 family, suggesting a connection between phospholipase C-derived signals and a proliferative cascade involving Ras (9).A 75-kDa inositol polyphosphate 5-phosphatase was initially identified in human platelet lysates (10). cDNAs encoding the enzyme have been isolated from human cDNA libraries (11,12). When expressed in COS cells, it shows Ins(1,4,5)P 3 , Ins(1,3,4,5)P 4 , PtdIns(4,5)P 2 , and PtdIns(3,4,5)P 3 5-phosphatase activities (13,14). A protein identified due to its deficiency in the Lowe's oculocerebrorenal syndrome has been shown to be homologuous to the 75-kDa inositol polyphosphate 5-phosphatase (15). Expression of a truncated form of the protein demonstrates Ins(1,4,5)P 3 , Ins(1,3,4,5)P 4 , and PtdIns(4,5)P 2 5-phosphatase activities (13). In brain, Type I 43-kDa Ins(1,4,5)P 3 5-phosphatase is the major enzyme hydrolyzing the calcium-mobilizing second messenger Ins(1,4,5)P 3 . It hydrolyzes both Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 with higher affinity for Ins(1,3,4,5)P 4 but lower velocity (16). PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 are not substrates (14,17). cDNAs encoding Type I Ins(1,4,5)P 3 5-phosphatase have been isolated from several dog and human cDNAs libraries (18 -20).Arginyl residues are known to act as anionic binding sites in proteins and may thus assist in the binding of substrates or enzyme catalysis. Covalent and irreversible modification with amino acid specific reagents has been used successfully to identify lysyl or arginyl residues in the substrate binding domain in many enzymes, such as tyrocidine synthetase 1 (21), Ca 2ϩ /ATPase (22), 6-phosphofructo-2-kinase (23), and Ins(1,4,5)P 3 3-kinase (24). In addition, two arginyl residues were shown using site-directed mutagenesis to be critical to bind the C-2 phospho group of fructose 2,6-bisphosphate in rat * This work was supported by grants of the FRSM, Boehringer Ingelheim, and the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Federal Service for Science, Technology and Culture. The costs of publication of this article ...

Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways.