PMID- 10441130
OWN - NLM
STAT- MEDLINE
DCOM- 19990908
LR  - 20131121
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 38
IP  - 32
DP  - 1999 Aug 10
TI  - Identification of the sites phosphorylated by cyclic AMP-dependent protein kinase
      on the beta 2 subunit of L-type voltage-dependent calcium channels.
PG  - 10361-70
AB  - Voltage-dependent L-type calcium (Ca) channels are heteromultimeric proteins that
      are regulated through phosphorylation by cAMP-dependent protein kinase (PKA). We 
      demonstrated that the beta 2 subunit was a substrate for PKA in intact cardiac
      myocytes through back-phosphorylation experiments. In addition, a heterologously 
      expressed rat beta 2a subunit was phosphorylated at two sites in vitro by
      purified PKA. This beta 2a subunit contains two potential consensus sites for
      PKA-mediated phosphorylation at Thr164 and Ser591. However, upon mutation of both
      of these residues to alanines, the beta 2a subunit remained a good substrate for 
      PKA. The actual sites of phosphorylation on the beta 2a subunit were identified
      by phosphopeptide mapping and microsequencing. Phosphopeptide maps of a
      bacterially expressed beta 2a subunit demonstrated that this subunit was
      phosphorylated similarly to the beta 2 subunit isolated from heart tissue and
      that the phosphorylation sites were contained in the unique C-terminal region.
      Microsequencing identified three serine residues, each of which conformed to
      loose consensus sites for PKA-mediated phosphorylation. Mutation of these
      residues to alanines resulted in the loss of the PKA-mediated phosphorylation of 
      the beta 2a subunit. The results suggest that phosphorylation of the beta 2a
      subunit by PKA occurs at three loose consensus sites for PKA in the C-terminus
      and not at either of the two strong consensus sites for PKA. The results also
      highlight the danger of assuming that consensus sites represent actual sites of
      phosphorylation. The actual sites of PKA-mediated phosphorylation are conserved
      in most beta 2 subunit isoforms and thus represent potential sites for regulation
      of channel activity. The sites phosphorylated by PKA are not substrates for
      protein kinase C (PKC), as the mutated beta 2 subunits lacking PKA sites remained
      good substrates for PKC.
FAU - Gerhardstein, B L
AU  - Gerhardstein BL
AD  - Department of Molecular Pharmacology and Biological Chemistry, Northwestern
      University Medical School, Chicago, Illinois 60611, USA.
FAU - Puri, T S
AU  - Puri TS
FAU - Chien, A J
AU  - Chien AJ
FAU - Hosey, M M
AU  - Hosey MM
LA  - eng
GR  - R01 HL23306/HL/NHLBI NIH HHS/United States
GR  - T32-DK07169/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Cacnb2 protein, rat)
RN  - 0 (Calcium Channels)
RN  - 0 (Calcium Channels, L-Type)
RN  - 0 (Peptide Fragments)
RN  - 0 (Phosphopeptides)
RN  - EC 2.7.11.11 (Cyclic AMP-Dependent Protein Kinases)
RN  - OS382OHJ8P (Cyanogen Bromide)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Binding Sites/genetics
MH  - Calcium Channels/*chemistry/genetics/*metabolism
MH  - *Calcium Channels, L-Type
MH  - Cell Line
MH  - Consensus Sequence/genetics
MH  - Cricetinae
MH  - Cyanogen Bromide/chemistry
MH  - Cyclic AMP-Dependent Protein Kinases/*chemistry/genetics/*metabolism
MH  - *Ion Channel Gating/genetics
MH  - Molecular Sequence Data
MH  - Myocardium/cytology/enzymology/metabolism
MH  - Peptide Fragments/chemistry/genetics
MH  - Peptide Mapping
MH  - Phosphopeptides/chemistry
MH  - Phosphorylation
MH  - Rabbits
EDAT- 1999/08/11 00:00
MHDA- 1999/08/11 00:01
CRDT- 1999/08/11 00:00
PHST- 1999/08/11 00:00 [pubmed]
PHST- 1999/08/11 00:01 [medline]
PHST- 1999/08/11 00:00 [entrez]
AID - 10.1021/bi990896o [doi]
AID - bi990896o [pii]
PST - ppublish
SO  - Biochemistry. 1999 Aug 10;38(32):10361-70. doi: 10.1021/bi990896o.