PtdIns(3,4,5) P 3 and PtdIns(3,4) P 2 are major signalling molecules in mammalian cell biology. PtdIns(3,4) P 2 can be produced by PI3Ks [PI (phosphoinositide) 3-kinases], but also by PI 5-phosphatases including SHIP2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2]. Proteomic studies in human cells revealed that SHIP2 can be phosphorylated at more than 20 sites, but their individual function is unknown. In a model of PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null astrocytoma cells, lowering SHIP2 expression leads to increased PtdIns(3,4,5) P 3 levels and Akt phosphorylation. MS analysis identified SHIP2 phosphosites on Ser 132 , Thr 1254 and Ser 1258 ; phosphotyrosine-containing sites were undetectable. By immunostaining, total SHIP2 concentrated in the perinuclear area and in the nucleus, whereas SHIP2 phosphorylated on Ser 132 was in the cytoplasm, the nucleus and nuclear speckles, depending on the cell cycle stage. SHIP2 phosphorylated on Ser 132 demonstrated PtdIns(4,5) P 2 phosphatase activity. Endogenous phospho-SHIP2 (Ser 132 ) showed an overlap with PtdIns(4,5) P 2 staining in nuclear speckles. SHIP2 S132A was less sensitive to C-terminal degradation and more resistant to calpain as compared with wild-type enzyme. We have identified nuclear lamin A/C as a novel SHIP2 interactor. We suggest that the function of SHIP2 is different at the plasma membrane where it recognizes PtdIns(3,4,5) P 3 , and in the nucleus where it may interact with PtdIns(4,5) P 2 , particularly in speckles.

IRBIT is an IP 3 R [IP 3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP 3 for binding to the IP 3 R. Phosphorylation of IRBIT is essential for the interaction with the IP 3 R. The unique N-terminal region of IRBIT, residues 1–104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo . IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser 68 of IRBIT. Phosphorylation of Ser 68 was required for subsequent phosphorylation of Ser 71 and Ser 74 , but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser 71 and Ser 74 were sufficient to enable inhibition of IP 3 binding to the IP 3 R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP 3 R. This pinpoints PP1 as a key player in the regulation of IP 3 R-controlled Ca 2+ signals.

The widely held view that SLN (sarcolipin) would be the natural inhibitor of SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca 2+ -ATPase 1), and PLB (phospholamban) its counterpart for SERCA2 inhibition is oversimplified and partially wrong. The expression of SLN and PLB mRNA and protein relative to SERCA1 or SERCA2 was assessed in ventricle, atrium, soleus and EDL (extensor digitorum longus) of mouse, rat, rabbit and pig. SLN protein levels were quantified by means of Western blotting using what appears to be the first successfully generated antibody directed against SLN. Our data confirm the co-expression of PLB and SERCA2a in cardiac muscle and the very low levels (in pig and rabbit) or the absence (in rat and mouse) of PLB protein in the slow skeletal muscle. In larger animals, the SLN mRNA and protein expression in the soleus and EDL correlates with SERCA1a expression, but, in rodents, SLN mRNA and protein show the highest abundance in the atria, which are devoid of SERCA1. In the rodent atria, SLN could therefore potentially interact with PLB and SERCA2a. No SLN was found in the ventricles of the different species studied, and there was no compensatory SLN up-regulation for the loss of PLB in PLB −/− mouse. In addition, we found that SLN expression was down-regulated at the mRNA and protein level in the atria of hypertrophic hearts of SERCA2 b / b mice. These data suggest that superinhibition of SERCA by PLB-SLN complexes could occur in the atria of the smaller rodents, but not in those of larger animals.

We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA ( p hospho t yrosyl p hosphatase a ctivator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn 2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.

About 24 intrinsic neurosecretory neurons within the pericardial organs (POs) of the crab Carcinus maenas produce a novel crustacean hyperglycaemic hormone (CHH)-like peptide (PO-CHH) and two CHH-precursor-related peptides (PO-CPRP I and II) as identified immunochemically and by peptide chemistry. Edman sequencing and MS revealed PO-CHH as a 73 amino acid peptide (8630Da) with a free C-terminus. PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence, positions 1–40, but the remaining sequence, positions 41–73 or 41–72, differs considerably. PO-CHH may have different precursors, as cDNA cloning of PO-derived mRNAs has revealed several similar forms, one exactly encoding the peptide. All PO-CHH cDNAs contain a nucleotide stretch coding for the SG-CHH 41–76 sequence in the 3′-untranslated region (UTR). Cloning of crab testis genomic DNA revealed at least four CHH genes, the structure of which suggest that PO-CHH and SG-CHH arise by alternative splicing of precursors and possibly post-transcriptional modification of PO-CHH. The genes encode four exons, separated by three variable introns, encoding part of a signal peptide (exon I), the remaining signal peptide residues, a CPRP, the PO-CHH 1–40 /SG-CHH 1–40 sequences (exon II), the remaining PO-CHH residues (exon III) and the remaining SG-CHH residues and a 3′-UTR (exon IV). Precursor and gene structures are more closely related to those encoding related insect ion-transport peptides than to penaeid shrimp CHH genes. PO-CHH neither exhibits hyperglycaemic activity in vivo , nor does it inhibit Y-organ ecdysteroid synthesis in vitro . From the morphology of the neurons it seems likely that novel functions remain to be discovered.